IRLF 


B    3 


flEfi 


MAY  27  1914 


A  STUDY  IN  CEREAL  RUSTS: 


PHYSIOLOGICAL  RACES 


A  THESIS  SUBMITTED  TO  THE  FACULTY 

OF 
THE  GRADUATE  SCHOOL 

OF 
THE  UNIVERSITY  OF  MINNESOTA 

BY 

ELVIN  CHARLES  STAKMAN 
IN  PARTIAL  FULFILMENT  OF  THE  REQUIREMENTS 

FOR  THE  DEGREE  OF 
DOCTOR  OF  PHILOSOPHY 


MINNEAPOLIS,   MINN. 
JUNE  1913 


A  STUDY  IN  CEREAL  RUSTS: 

PHYSIOLOGICAL  RACES 


A  THESIS  SUBMITTED  TO  THE  FACULTY 

OF 
THE  GRADUATE  SCHOOL 

OF 
THE  UNIVERSITY  OF  MINNESOTA 

BY 

ELVIN  CHARLES  STAKMAN 
IN  PARTIAL  FULFILMENT  OF  THE  REQUIREMENTS 

FOR  THE  DEGREE  OF 
DOCTOR  OF  PHILOSOPHY 


MINNEAPOLIS,  MINN. 
JUNE  1913 


n 


* 


SB* 


TABLE  OF  CONTENTS 

Page 

Part  I.     Biologic  Forms   7 

Historical    7 

Experimental    10 

Methods 10 

Experiments  with  Puccinia  graminis  hordei 12 

General  statement   12 

Inoculations  on  rye 12 

Inoculations  on  oats 13 

Inoculation  after  the  use  of  anesthetics 13 

Inoculations  on  wheat 14 

Summary  of  inoculations  with  Puccinia  graminis  hordei.  . .  15 

Experiments  with  Puccinia  graminis  avenae 15 

General  statement 15 

Inoculations  on  rye 15 

Inoculations  under  ordinary  conditions 15 

Effect  of  high  fertilization 16 

Effect  of  anesthetics 16 

Effect  of  leaf  injury 16 

Inoculations  on  wheat 17 

Inoculations  under  ordinary  conditions 17 

Effect  of  anesthetics  17 

Effect  of  manure 18 

Effect  of  leaf  injury   18 

Summary  of  inoculations  on  wheat. 18 

Inoculations  on  barley 19 

Inoculations  under  ordinary  conditions 19 

Inoculations  after  exposure  to  anesthetics 19 

Summary    of    inoculations    made    with    Puccinia    graminis 

avenae   19 

Experiments  with  Puccinia  graminis  secalis 20 

Inoculations  on  wheat 20 

Inoculations  on  oats 20 

Inoculations  on  barley   20 

Inoculations  on  einkorn  21 

Experiments  with  Puccinia  graminis  tritici 21 

Inoculations  on  barley 21 

Inoculations  on  rye 21 

Inoculation  under  ordinary  conditions.  .                              ,  21 


293420 


6  CONTENTS 

Inoculation  after  exposure  to  ether %  21 

Inoculations  on  oats  , . 21 

Inoculations  under  ordinary  conditions 21 

Inoculation  after  exposure  to  ether 22 

Inoculations  on  oats  and  rye  after  the  use  of  barley  as  a 

"bridging  form" 22 

Summary  of  experiments  with  Puccinia  graminis  tritici. ...  22 

Effect  of  the  aecidial  stage  on  biologic  forms 23 

General  statement  23 

Experiments  in  1912 < 23 

Experiments  in  1913 24 

Adaptation  of  biologic  forms  to  new  hosts 25 

Summary  of  Part  1 27 

Part  II.    Rust-resistant  varieties  of  wheat 28 

Historical 28 

Forms  which  are  resistant 29 

Field  observations  29 

Experimental    '. 30 

Greenhouse  trials 30 

Inoculations  on  resistant  forms 32 

Metabolism  of  the  host  and  rust  resistance 33 

Effect  of  water  content  of  soil 34 

Effect  of  fertilizers  - 36 

The  nature  of  resistance 40 

Histological  details  of  infection 42 

Infection  of  Minnesota  No.  163 42 

Infection  of  Khapli 44 

The  course  of  infection  in  other  resistant  forms 45 

Minnesota  No.  163  inoculated  with  Puccinia  graminis  avenae  46 

Summary  of  Part  II 48 

Bibliography  ...  * 50 

Explanation  of  Plates 55 


A  STUDY  IN  CEREAL  RUSTS 
PHYSIOLOGICAL  RACES 

PART  I.    BIOLOGIC  FORMS 

HISTORICAL 

Cereal  rusts  were  known  as  destructive  plant  pests  by  the  ancients. 
Observations  were  made  on  the  effect  of  weather  and  location  on  their 
prevalence.  The  comparative  susceptibility  of  the  various  cereals 
received  some  attention.  Theophrastus  speaks  of  the  varying  suscepti- 
bility of  different  cereals,  as  does  Pliny,  who  says  that  barley  is  less 
likely  to  rust  than  are  other  grains.  In  modern  times  many  observa- 
tions have  been  made,  but  many  of  these  were  only  incidental. 

Dietel  (1887)  calls  attention  to  a  certain  amount  of  morphological 
and  physiological  variation  in  rust  fungi,  but  does  not  definitely  estab- 
lish the  fact  that  there  are  distinct  biologic  forms.  Previous  to  his 
time  Puccinia  graminis  Pers.  was  considered  a  single  species  found 
on  cereals  and  various  grasses.  However,  in  1894  Eriksson  (1894-1) 
showed  that,  although  the  morphology  of  the  fungus  on  the  different 
cereal  hosts  varied  but  slightly,  there  was  a  distinct  specialization  in 
parasitism.  He  therefore  divided  the  different  species  of  rusts  into 
subdivisions  which  he  termed  "formae  speciales."  Puccinia  graminis, 
the  only  species  extensively  used  in  the  present  investigation,  he  di- 
vided into  five  formae  speciales,  two  of  which  he  mentioned  as  being 
sharply  demarcated  and  the  remainder  as  being  probably  also  distinct. 

All  of  these  forms  of  Puccinia  graminis  were  capable  of  produc- 
ing aecidia  on  various  species  of  Berberis.  It  therefore  occurred  to 
Eriksson  that  perhaps  his  formae  speciales  would  be  equalized  when 
grown  upon  the  alternate  host.  This,  however,  he  found  not  to  be  the 
case.  He  concluded  that  the  forms  were  physiologically  distinct  even 
when  grown  upon  Berberis.  In  fact  he  was  led  to  believe  that  they 
became  more  firmly  fixed  after  a  period  on  the  barberry. 

This  discovery  stimulated  much  research  concerning  the  physiolog- 
ical relationships  of  the  Uredineae.  In  the  United  States  Hitchcock 
and  Carleton  (1894)  made  observations  at  about  the  same  time  that 

1 


8          .    .  .  tS  fct.  .- .  -4  STUDY-  JN  CEREAL  RUSTS 

Eriksson  was  making  his  in  Sweden.  Their  conclusions  pointed  to 
the  fact  that  there  was  not  much  danger  of  rust  from  one  cereal  infect- 
ing another.  The  same  phenomenon  was  observed  in  rusts  on  a  wide 
variety  of  hosts.  Magnus  (1894  and  1895),  Rostrup  (1894),  Klebahn 
(1896),  Dietel  (1899),  Ward  (1901),  Bandi  (1903),  and  others  firmly 
established  the  fact  that  this  specialization  of  parasitism  was  quite 
common  in  the  various  rusts.  To  the  formae  speciales  of  Eriksson 
various  names  were  applied.  Schroeter  (see  Magnus  1894,  p.  360) 
called  them  Schwester-Arten,  and  Rostrup  (1894,  p.  40)  called  them 
biologische  Arten,  while  Hitchcock  and  Carleton  (1894)  referred  to 
them  as  physiological  races. 

Neger  (1902)  found  evidences  of  a  similar  condition  among  the 
Erysiphaceae.  Marchal  (1902)  conducted  a  large  number  of  cross-inoc- 
ulations with  Erysiphe  graminis  and  concluded  that  there  was  a  fairly 
large  number  of  "races  specialisees."  These  did  not  differ  essentially 
in  any  morphological  character.  He  showed  (1903)  and  Salmon 
(1903-2)  later,  showed  that  the  ascospores  behaved  in  the  same  way 
with  regard  to  this  specialization  of  parasitism  as  did  the  conidia. 
Reed  (1905)  has  shown  that  there  may  be  more  than  one  physiological 
race  upon  a  single  genus  of  host  plant. 

Magnus  was  one  of  the  first  to  try  to  explain  the  phenomenon  of 
specialization  of  parasitism.  He  distinguishes .  (1894,  p.  366)  be- 
tween "Gewohnheitsrassen"  or  adaptive  races  and  biologic  forms. 
The  former  name  he  applies  to  such  forms  as  merely  show  difference 
in  infection  power,  while  those  which  are  fixed  he  calls  biologic  forms. 
Long  association  with  one  host  plant,  he  says,  may  bring  the  develop- 
ment from  Gewohnheitsrassen  to  biologic  forms.  This  he  showed 
(1895)  to  be  true  not  only  of  rust  fungi  but  of  others  as  well.  Dietel 
(1899)  expressed  a  somewhat  similar  opinion,  his  idea  being  that 
formerly  a  given  species  attacked  a  variety  of  hosts,  but  that  it  be- 
came more  and  more  specialized  to  form  first  Gewohnheitsrassen  and 
then  biologic  forms.  Eriksson  (1902,  p.  657)  says  that  rust  forms  adapt 
themselves.  Where  a  certain  host  is  present  in  large  numbers,  and 
climatic  conditions  are  favorable,  changes  take  place  in  favor  of  the 
new  host.  These  changes  are  expressed  not  only  in  the  vitality  of  the 
fungus  but  also  in  a  higher  degree  of  systematic  firmness.  The  new 
rust  form,  he  says,  becomes  separated  from  its  sister  forms  of  parallel 
origin  and  becomes  "scharf  fixiert."  His  conclusion  is,  "Das  Phanomen 
der  Spezialisierung  steht  nicht  langer  da  als  der  Exponent  eines  dem 
Schmarotzer  innewohnenden,  launenhaften  und  unerklarlichen  Triebes, 
iieue  Formen  zu  produzieren.  Dieser  Trieb  wird  durch  die  umge- 
benden  Verhaltnisse — die  vegetative  Unterlage  und  das  Klima, — 
unter  denen  der  Parasit  lebt,  in  eine  bestimmte  Richtung  geleitet." 
This,  Eriksson  (1902,  pp.  606  and  654)  thinks,  accounts  for  the  fact 


BIOLOGIC  FORMS  9 

that  the  specialization  has  taken  a  different  course  in  Sweden  from 
that  it  has  followed  in  the  United  States.  The  most  widely  grown 
crops  would  naturally  be  the  ones  on  which  the  particular  biologic 
forms  adapted  to  them. would  attain  their  highest  development.  There- 
fore, the  fact  that  a  rust  shows  particular  relationships  in  one  country 
does  not  by  any  means  preclude  the  possibility  of  a  quite  different  set 
of  relationships  in  another  country. 

The  idea  that  the  fungus  changes  its  habits  as  a  result  of  environ- 
ment is  substantiated  by  many  observations.  Perhaps  an  extreme  case 
of  such  a  tendency  is  found  in  the  life  history  of  Puccinia  graminis  in 
Australia.  McAlpine  (1906,  p.  21)  states  that  the  teleutospores  seem 
unable  to  infect  the  barberry  in  Australia,  and,  according  to  his  obser- 
vations, the  fungus  is  quite  rapidly  being  reduced  to  a  reproduction 
by  uredospores  only.  This  is  accounted  for  by  the  absence  of  the  bar- 
berry. 

Eriksson  (1896,  p.  339)  shows  that  closely  related  host  forms 
are  somewhat  similar  in  their  relation  to  rust.  He  says,  however,  that 
the  taxonomic  relationship  of  host  plants  does  not  entirely  determine 
the  specialization  of  the  rust  form.  Ward  (1901)  in  his  work  on  the 
rust  (Puccinia  dispersa)  of  the  bromes  states  that  the  closeness  of  re- 
lationship of  hosts  is  the  determining  factor  in  the  ability  of  the  rust 
to  pass  successfully  from  one  host  plant  to  another.  Freeman  (1902) 
also  concluded  that  the  farther  removed  a  species  of  Bromus  was  tax- 
onomically  from  the  plant  serving  as  a  host  for  the  rust  the  less  prob- 
ability there  was  of  infection.  Ward  showed  further  (1903)  that  some 
forms  of  bromes  might  act  as  bridging  species  in  enabling  the  rust  to 
pass  indirectly  from  one  group  of  bromes  to  another,  although  direct 
transfer  was  impossible.  Salmon  (1904)  showed  that  the  same  thing 
was  true  of  Erysiphe  graminis  D.  C.  Freeman  and  Johnson  (1911) 
have  found  that  barley  can  act  as  a  bridging  form  enabling  Puccinia 
graminis  to  increase  its  range  of  infection  power.  Salmon  (1904  and 
1905)  showed  that  the  range  of  infection  possibility  of  Erysiphe 
graminis  forms  may  also  be  increased  under  certain  cultural  conditions. 
By  injuring  leaves  and  subjecting  plants  to  heat  and  anesthetics  he 
was  able  to  infect  normally  immune  forms. 

The  conception  of  a  biologic  form,  then,  is  that  it  represents  a 
tendency  toward  adaptation.  This  tendency  may  be  due  to  various 
causes,  the  evidence  being  that  it  depends  largely  on  the  availability 
of  host  species.  Hitchcock  and  Carleton  (1894),  Carleton  (1899),  and 
Freeman  and  Johnson  (1911)  investigated  quite  thoroughly  the  mat- 
ter of  biologic  forms  of  Puccinia  graminis  in  the  United  States.  Free- 
man and  Johnson  (1911,  p.  27)  give  the  following  as  the  biologic 
forms  of  this  rust  in  the  United  States : 

P.     graminis  tritici  (stem  rust  of  wheat)  on  wheat  and  barley. 


10  A  STUDY  IN  CEREAL  RUSTS 

P.  graminis  hordei  (stem  rust  of  barley)  on  barley,  wheat,  and 
rye. 

P.     graminis  secalis  (stem  rust  of  rye)  on  rye  and  barley. 

P.     graminis  avenae  (stem  rust  of  oats)  on  oats. 

If  these  forms  are  merely  adaptations  it  ought  to  be  possible  to 
change  their  parasitic  tendencies  by  restricting  or  changing  their  en- 
vironment. It  ought  to  be  possible  to  break  down  the  biologic  forms 
under  conditions  abnormal  for  host  or  parasite.  Various  methods 
have  been  tried  in  attempting  to  break  down  the  specialization  of  para- 
sitism of  different  fungi.  Salmon's  work  along  this  line  on  the  Ery- 
siphaceae  has  already  been  mentioned.  Ray  (1903)  states  that  by 
subjecting  maize  to  ether  vapor  and  then  inoculating  it  with  spores  of 
Ustilago  zeae,  the  resulting  infection  was  much  more  virulent  than 
that  on  plants  not  so  treated. 

Comparatively  little  work  of  this  nature  has  been  done  with  Puc- 
cinia  graminis.  The  fact  that  physiological  races  of  rusts  behave  dif- 
ferently under  different  conditions  has  been  known  for  some  time. 
Much  of  the  information  was,  however,  gathered  from  incidental 
observations.  In  breeding  wheats  for  the  purpose  of  obtaining  rust- 
resistant  forms  it  would  be  very  helpful  to  be  able  to  correlate  certain 
characters  with  rust  resistance.  For  this  reason  this  phase  of  the 
question,  from  both  practical  and  scientific  points  of  view,  is  of  much 
importance.  The  same  is  true  of  physiological  races.  It  is  important 
to  know  if  they  can  be  broken  down  by  means  of  a  high  degree  of  soil- 
fertilization,  if  they  become  generalized  by  growing  on  the  alternate 
host,  and  if  they  adapt  themselves  readily  to  new  hosts.  The  present 
investigation  was  therefore  undertaken  with  the  object  of  determining 
the  possibility  of  developing  and  breaking  down  physiological  races 
and  of  obtaining  definite  information  concerning  the  factors  influencing 
varying  resistance  in  immune  or  semi-immune  varieties  of  wheat. 

EXPERIMENTAL 

METHODS 

The  rusts  used  in  making  inoculations  were  obtained  originally 
from  their  respective  hosts  in  the  fields  at  University  Farm,  St.  Paul, 
Minnesota.  They  were  then  artificially  transferred  to  plants  growing 
in  the  greenhouse.  Transfers  were  made  to  new  plants  about  once 
every  three  weeks  until  the  rust  had  been  confined  to  its  own  host  for 
at  least  twelve  successive  transfer  generations.  In  nearly  all  the  exper- 
iments with  biologic  forms  the  rust  had  been  confined  to  its  own  host 
for  at  least  twenty  generations,  thus  giving  assurance  that  it  was  the 
particular  biologic  form  desired. 

The  seeds  of  the  host  plants  were  planted  in  rich  loam  soil  in  four- 


BIOLOGIC  FORMS  11 

inch  clay  pots.  Only  ten  plants  were  left  in  each  pot  and  the  first  leaf 
of  each  was  inoculated  when  six  or  seven  days  old.  The  plants  were 
trimmed  whenever  necessary  so  as  to  leave  only  the  one  inoculated  leaf 
on  each  plant.  Fresh,  viable  uredospores  were  used  for  inoculations 
except  where  otherwise  specified.  The  spores  were  put  on  the  leaves 
with  a  flat  inoculating  needle  which  had  been  previously  moistened 
in  order  that  the  spores  might  better  adhere  to  the  leaf  surface.  The 
pots  were  then  placed  in  shallow  pans  filled  with  water,  or  on  wet 
sand,  and  covered  with  bell  jars  for  forty-eight  hours.  In  nearly  all 
cases  a  fine  film  of  moisture  covered  the  leaves  during  a  considerable 
part  of  the  time  that  they  were  under  the  jars.  This,  together  with  a 
moderate  temperature,  made  the  conditions  for  infection  ideal.  After 
the  removal  of  the  bell  jars  the  plants  were  kept  on  greenhouse  benches 
in  such  locations  as  to  reduce  to  the  minimum  the  danger  of  accidental 
infection. 

The  grains  used  were  the  following,  the  numbers,  except  where 
specified,  being  Grain  Investigation  numbers  of  the  United  States 
Department  of  Agriculture : 

Fife  wheat,  Minnesota  No.  163 

Velvet  Blue  Stem  wheat,  Minnesota  No.  169 

Minnesota  No.  188  wheat,  a  cross  between  White  Fife  and  Ladoga 
Fife 

Manchuria  barley,  Minnesota  No.  105 

Early  Gothland  oats,  Minnesota  No.  295 

Swedish  rye,  Minnesota  No.  2 

Kubanka  1516 — Nos.  8  and  9  pedigreed — Dickinson,  N.  Dak.,  1910 

Kubanka  2094 

Arnautka  288 

Arnautka  1431 

lumillo  1736  (1736-II-3  selected  at  Amarillo,  Tex.,  1910) 

Einkorn  (Triticum  monococcum)  2433 — Nos.  4,  6,  7,  and  8  pedi- 
greed—Dickinson, N.  Dak.,  1910 

Emmer  (Triticum  dicoccum)   1522 

Khapli  (an  Indian  Emmer) 

Of  these  the  Kubankas,  Arnautka,  and  lumillo  are  varieties  of 
Triticum  durum.  The  durums  generally  have  the  reputation  of  being 
more  resistant  to  rusts  than  are  the  ordinary  wheats  (Carleton  1905,  p. 
9).  Einkorn,  also,  is  quite  resistant  while  the  emmers  vary  greatly, 
one  used  in  this  work,  G.  I.  No.  1522,  not  being  very  resistant.  Khapli 
is  an  emmer  obtained  from  India  by  E.  C.  Johnson,  formerly  Cereal 
Pathologist  in  the  office  of  Grain  Investigations,  United  States  Depart- 
ment of  Agriculture.  It  has  no  Grain  Investigation  number.  It  is  the 
most  resistant  of  all  the  forms  used. 


12  A  STUDY  IN  CEREAL  RUSTS 

EXPERIMENTS  WITH  PUCCINIA  GRAMINIS  HORDEI 

GENERAL  STATEMENT 

Freeman  and  Johnson  (1911,  p.  20)  state  that  the  barley  stem 
rust  is  more  versatile  than  any  of  the  other  biologic  forms  of  the  cereal 
rusts,  and,  further,  that  the  range  of  infection  of  a  given  form  is  in- 
creased after  having  been  transferred  to  barley.  The  results  of  the 
author,  in  general,  agree  with  those  of  Freeman  and  Johnson,  although 
the  percentages  of  infection  are  in  some  cases  quite  different. 

INOCULATIONS  ON   RYE 

In  the  first  series  20  leaves  were  inoculated,  after  which  the  pots 
were  kept  under  bell  jars  for  48  hours.  In  8  days  very  distinct  signs 
of  infection  began  to  appear  and  on  the  tenth  day  distinct  pustules 
were  quite  numerous.  Eventually  16  leaves  out  of  18  showed  distinct 
pustules.  Two  of  the  leaves  had  been  killed.  Very  evidently  not  all 
of  the  mycelial  wefts  developed  pustules,  since  pustules  were  often 
intermingled  with  typical,  yellowish  rust  flecks  and  green  islands.  The 
pustules  were  in  all  cases  small,  most  of  them  less  than  a  millimeter  in 
size,  but  they  were  fairly  numerous.  There  were  some  fairly  large 
areas  in  which  no  pustules  developed,  but  which  contained  a  large 
number  of  green  islands. 

In  the  second  series  40  leaves  were  inoculated,  10  of  which  eventu- 
ally produced  pustules.  Of  the  remaining  30  leaves  29  were  very 
strongly  flecked  in  such  a  manner  as  to  show  conclusively  that  infec- 
tion had  taken  place  although  no  pustules  had  been  formed. 

The  results  of  both  series  indicate  that  rye  is  easily  infected  by 
the  stem  rust  from  barley.  Out  of  58  leaves,  26  were  infected,  indi- 
cating greater  ease  of  infection  than  in  case  of  the  strain  used  by 
Freeman  and  Johnson  (1911,  p.  19)  in  their  experiments.  The  29 
leaves  which  are  indicated  as  having  been  strongly  flecked  would  at 
first  sight  have  been  counted  as  having  pustules.  However,  close 
examination  showed  that  there  were  no  ruptured  pustules. 

Although  a  fairly  high  percentage  of  successful  infections  can  be 
obtained,  rye  is  by  no  means  a  congenial  host  for  Puccinia  graminis 
from  barley.  This  is  shown  by  the  fact  that  the  pustules  are  always 
small.  Long  areas  of  the  leaf  may  be  killed  and  in  this  area  there  may 
be  many  unruptured  pustules.  Often  an  area  extending  across  the 
entire  leaf  and  one  centimeter  or  more  in  length  may  be  completely 
killed.  (See  Plate  I,  A.)  This  may  not  contain  a  single  pustule,  but 
close  examination  reveals  the  fact  that  there  are  many  green  islands, 
some  of  which  have  a  yellowish  tinge  in  the  center.  These  latter  look 
very  much  like  unruptured  pustules.  Histological  examination  re- 
veals the  fact  that  the  mycelium  has  spread,  the  host  cells  have  died, 
and  wefts  of  mycelium  have  formed,  either  directly  under,  or  a  slight 


BIOLOGIC  FORMS  13 

distance  below,  the  epiderm.  These  wefts  may  send  up  a  few  hyphae 
which  resemble  those  normally  producing  spores.  In  some  cases  a 
few  small,  abortive  spores  are  formed,  while  in  other  cases,  none  are 
produced. 

This  phenomenon  very  closely  parallels  that  occurring  in  some  of 
the  resistant  forms  of  wheat.  Attention  will  be  called  to  this  more  in 
detail  later  on.  It  will  be  sufficient  here  to  emphasize  the  fact  that 
rye,  when  inoculated  with  Puccinia  graminis  spores  from  rye,  does  not 
show  any  of  these  dead  areas  (see  Plate  I,  B),  the  leaves  remaining 
green  and  producing  pustules  vigorously.  When  the  spores,  however, 
are  taken  from  barley,  infection  takes  place,  but  there  is  not  such  a  per- 
fect relationship  set  up  between  host  and  parasite  as  to  enable  both  to 
live  and  thrive  for  a  long  time. 

INOCULATIONS   ON    OATS 

The  first  attempt  to  infect  oats  with  stem  rust  from  barley  failed 
absolutely.  For  this  reason  the  greatest  precautions  were  taken  to 
furnish  optimum  conditions  for  germination  of  spores  and  develop- 
ment of  the  mycelium.  Of  104  leaves  inoculated  at  various  times,  not 
one  produced  pustules.  Of  this  number  8  were  slightly  flecked,  but 
showed  no  tendency  to  form  pustules.  In  fact,  the  flecks  were  ex- 
tremely small  and  very  few  occurred  on  each  leaf.  In  some  cases 
there  was  but  a  single  fleck.  These  might  quite  easily  have  escaped 
detection  on  some  of  the  leaves  had  they  not  been  invariably  situated 
in  the  inoculated  area.  Their  position  in  this  area  indicates  quite 
strongly  that  they  were  true  rust  flecks.  In  these  areas  the  tissues  of 
the  leaves  seemed  to  have  been  killed  outright.  However,  the  mycelium 
must  have  been  very  restricted  in  its  development  since  the  diameter 
of  a  spot  never  exceeded -one  millimeter. 

Inoculation  after  the  use  of  anesthetics — 

In  the  first  trial  two  pots  of  oats  were  exposed  to  the  fumes  of 
ether  for  15  minutes.  Immediately  after  tlie  exposure  they  were 
inoculated  with  fresh  spores  of  Puccinia  graminis  from  barley  and 
then  placed  under  a  bell  jar  in  a  shallow  pan  of  water.  Two  pots 
were  exposed  to  chloroform  fumes  for  an  equal  length  of  time  and 
inoculated.  At  the  same  time  25  leaves  were  inoculated  under  normal 
conditions,  as  a  check. 

Of  those  leaves  exposed  to  ether,  4  out  of  a  total  of  20  developed 
slight  flecks  but  no  pustules ;  neither  were  there  any  unruptured  pus- 
tules, which  are  commonly  found  when  infection  is  not  normal.  Of 
those  exposed  to  chloroform  2  out  of  19  developed  pustules  in  12 
days.  These  pustules  were  very  small,  but  were  unquestionably  pus- 
tules of  stem  rust.  A  few  of  the  leaves  were  so  indistinctly  flecked 
that  it  was  doubtful  if  they  were  true  rust  flecks. 


14  A  STUDY  IN  CEREAL  RUSTS 

In  the  second  trial  plants  were  again  exposed  to  ether  and  chloro- 
form, but  the  time  was  increased  from  15  to  20  minutes.  An  attempt 
was  made  to  insure  conditions  for  successful  infection.  It  was  ob- 
served that  a  film  of  moisture  formed  on  the  leaves  at  night  and  the 
temperature  was  moderate,  insuring  successful  infection  if  that  was 
ordinarily  possible. 

Of  the  20  leaves  exposed  to  ether,  11  became  very  distinctly 
flecked,  but  there  were  no  evidences  whatever  of  successful  pustule 
formation.  In  the  chloroform  series  one  leaf  out  of  20  produced  a 
small  pustule  and  8  became  clearly  flecked.  -There  were  20  check 
leaves,  and,  of  these,  4  became  flecked.  The  flecking  was,  however, 
rather  indistinct. 

Out  of  a  total  of  193  inoculations  only  3  resulted  in  pustule-for- 
mation, and  these  only  after  exposure  to  chloroform.  Of  the  remain- 
der, 31  were  flecked,  but  this  flecking  was  not  always  sharp  on  the 
control  plants.  In  the  other  cases,  however,  it  was  distinct.  The  spots 
were  always  less  than  one  millimeter  in  diameter,  and  usually  much 
smaller.  It  appeared  that  this  small  area  of  the  leaf  had  been  killed, 
thus  preventing  the  further  spread  of  the  mycelium  and  precluding 
the  possibility  of  pustule  formation.  In  no  case  was  there  any  indica- 
tion whatever  that  large  areas  of  the  leaf  were  involved,  as  was  the 
case  when  rye  was  inoculated  with  the  rust  from  barley.  In  this  con- 
nection it  may  be  mentioned  that  Freeman  and  Johnson  (1911,  p.  19) 
were  able  to  get  7  successful  infections  out  of  a  total  of  35,  but  the 
pustules  were  always  very  small.  It  may  be  that  the  difference  is  due 
to  the  use  of  various  strains  of  the  rust.  The  fact  that  there  are  strains 
of  the  same  biologic  form  seems  to  be  quite  definitely  indicated.  In 
any  case  this  seems  to  furnish  an  example  of  rather  extreme  incom- 
patibility between  host  and  parasite.  In  some  cases  it  would  seem  that 
the  infection  threads  of  the  fungus  are  checked  almost  immediately ; 
or  that  they  may  gain  a  temporary  foothold,  only  to  kill  the  cells  upon 
which  they  depend  for  nourishment  and  then  develop  but  little  further. 

INOCULATIONS   ON    WHEAT 

There  is  no  question  but  that  the  stem  rust  from  barley,  in  this 
country,  usually  passes  to  wheat  with  practically  the  same  degree  of 
readiness  with  which  it  passes  to  barley.  In  the  trials  made  with  this 
form  this  appeared  to  be  the  case  whether  susceptible  or  resistant  forms 
of  wheat  were  used.  Inoculations  on  Arnautka,  Khapli,  and  emmer 
show  that  the  barley  rust  is  quite  as  capable  as  is  the  wheat  rust  of 
attacking  these  varieties. 

Pritchard  (1911-1,  pp.  181,  182)  cites  evidence  tending  to  show 
that  the  forms  on  wheat  and  barley  in  North  Dakota  are  distinct.  This 
is  not  the  case,  however,  with  the  strains  used  in  the  experiments  in 
Minnesota. 


BIOLOGIC  FORMS  15 

SUMMARY  OF  INOCULATIONS  WITH  PUCCINIA  GRAMINIS  HORDEI 

Wheat  and  barley  are  not  included  since  in  nearly  every  case  100 
per  cent  of  infection  results.    The  denominator  in  each  case  represents 
the  total  number  of  leaves  inoculated,  and  the  numerator,  the  number 
of  leaves  which  developed  pustules. 
Barley  rust  to: 

OatsT$T;  8  slightly  flecked 

Oats  after  exposure  to  ether    o  .  JEJ  flecked 

Oats  after  exposure  to  chloroform  -£-§  ;  8  flecked 
RyeJ-1;  29  very  strongly  flecked 

It  will  thus  be  seen  that  barley  rust  does  not  find  either  rye  or  oats 
a  congenial  host.  It  was  transferred  to  rye  much  more  easily  in  these 
experiments  than  in  those  reported  by  other  investigators  in  this  coun- 
try. Without  exposing  the  host  plants  to  anesthetics,  however,  no  suc- 
cessful infection  of  oats  was  obtained.  By  the  use  of  ether  and  chloro- 
form the  possibility  of  infection  was  somewhat  increased,  as  evidenced 
by  the  formation  of  pustules  on  a  small  percentage  of  leaves  and  the 
increased  percentage  of  flecked  leaves.  In  addition  to  this,  the  flecks 
were  much  more  distinct  than  those  on  the  check  plants. 

EXPERIMENTS  WITH  PUCCINIA  GRAMINIS  AVENAE 

GENERAL  STATEMENT 

This  rust  in  this  country  is  supposed  to  infect  no  cereal  except 
oats,  although  it  is  capable  of  infecting  a  number  of  grasses.  Carleton 
(1899,  p.  60)  was  unable  to  transfer  it  successfully  to  wheat,  barley, 
or  rye.  Eriksson  (1902,  p.  601)  mentions  it  as  being  found  on  oats 
and  18  species  of  grasses  in  Sweden.  Freeman  and  Johnson  (1911, 
p.  22),  however,  find  that  it  can  be  transferred  to  barley  also,  and  they 
report  that  Derr  succeeded  in  obtaining  direct  transfers  from  oats  to 
wheat  and  rye. 

INOCULATIONS  ON  RYE 

Since  Derr,  according  to  Freeman  and  Johnson  (1911,  p.  22), 
obtained  but  one  successful  infection  by  inoculating  rye  with  stem 
rust  of  oats,  a  large  number  of  trials  were  made  with  this  form,  espe- 
cially since  the  same  authors  (I.e.  p.  23)  assert  that  under  favorable 
conditions  these  can  undoubtedly  be  made. 

Inoculations  under  ordinary  conditions — 

The  results  of  inoculations  made  under  average  conditions  show 
clearly  that  it  is  possible  to  transfer  the  rust  from  oats  to  rye.  Al- 
though the  percentage  of  entirely  successful  infections^was  small,  the 


16  A  STUDY  IN  CEREAL  RUSTS 

appearance  of  inoculated  plants  would  lead  one  to  believe  that  the  rusl 
transfers  with  greater  ease  than  it  really  does.  Out  of  a  total  of  55 
leaves  inoculated  only  3  produced  pustules.  These  pustules,  although 
small,  were  very  distinct.  Of  the  remaining  leaves,  13  were  clearlv 
flecked.  It  is  the  appearance  of  these  so-called  flecked  leaves  which  is 
often  misleading.  When  looking  at  them  from  a  distance  they  some- 
times appear  to  be  quite  badly  rusted.  Closer  examination,  however, 
shows  that  there  are  no  pustules.  Sometimes  the  flecks  are  so  distinct 
as  to  suggest  the  appearance  of  unruptured  pustules. 

The  effect  of  high  fertilization — 

A  rich  loam  was  well  mixed  with  rich  barnyard  manure.  Rye 
was  planted  in  two  pots  and  the  leaves  inoculated  in  the  usual  manner. 
Evidences  of  infection  appeared  at  the  usual  time.  It  was  found  thai 
the  severity  of  infection  was  much  greater  than  that  on  plants  grown 
in  unfertilized  soil.  Of  a  total  of  18  leaves  9  developed  pustules  and 
the  other  9  were  distinctly  flecked.  The  areas  of  infection  on  these 
plants  were  much  larger  than  on  those  grown  in  ordinary  soil.  The 
leaf  tissues  were  very  clearly  killed,  sometimes  in  areas  a  centimeter 
long,  and  in  these  areas  small  pustules  appeared.  The  appearance 
was  very  characteristic  of  semi-normal  infection.  The  mycelium  spread 
fairly  well,  but  the  host  cells  were  killed  and  only  small  pustules  were 
developed. 

The  effect  of  anesthetics — 

Plants  were  inoculated  after  exposure  to  ether,  chloroform,  and 
nitrous  oxide  for  periods  ranging  in  different  experiments  from  5  to 
15  minutes.  The  difference  between  the  check  plants  and  those  ex- 
posed to  anesthetics  for  10  minutes  was  fairly  distinct.  This  was  not 
apparent  so  much  in  the  number  of  pustules  or  flecks,  but  rather  in  the 
sharpness  of  the  flecks.  The  following  were  the  results : 

After  exposure  to  ether -g^-;  26  flecked 
After  exposure  to  chloroform  -^;  13  flecked 
After  exposure  to  nitrous  oxide  -^-5-;  6  flecked 

It  will  thus  be  observed  that  in  point  of  numbers  the  difference 
between  the  success  of  these  infections  and  those  on  the  check  plants 
was  not  great.  However,  a  real  difference  in  severity  of  infection, 
although  not  remarkable,  did  exist.  On  the  other  hand  the  plants  in 
highly  fertilized  soil  developed  a  larger  number  of  pustules  and  also  a 
more  typical  infection. 

Effect  of  leaf  injury — 

A  number  of  leaves  were  injured,  just  previous  to  inoculation,  by 
being  punctured  in  many  places  with  a  sterilized  needle  point.  Spores 
were  then  placed  on  this  injured  area  in  larsfe  numbers.  The  epidermis 


BIOLOGIC  FORMS  17 

was  stripped  from  others  and  the  inoculations  then  made.  The  results 
were  not  different  from  those  obtained  on  check  plants.  Only  one 
pustule  developed  on  one  leaf  out  of  a  total  of  28.  Six  of  the  remain- 
ing leaves  were  flecked,  but  the  flecks  remained  small.  In  no  case  did 
the  virulence  of  infection  equal  that  on  plants  grown  in  highly  fertilized 
soil. 

The  stem  rust  of  oats,  then,  can  be  transferred  directly  to  rye. 
This  was  accomplished  most  easily  when  the  plants  were  grown  in 
heavily  manured  soil.  Exposing  them  to  various  anesthetics  before 
inoculation  seems  to  increase  the  virulence  of  infection  slightly,  while 
leaf  injury  had  no  apparent  effect.  In  the  various  trials,  under  differ- 
ent conditions,  27  out  of  236  leaves  developed  pustules  and  73  were 
flecked.  Under  conditions  which  might  exist  in  the  field  12  out  of  73 
leaves  produced  pustules  and  22  were  flecked. 

INOCULATIONS  ON   WHEAT 

The  stem  rust  of  oats  can  be  transferred  to  wheat  only  with  great 
difficulty.  Carleton  (1899,  p.  60)  did  not  succeed  in  obtaining  infec- 
tion in  his  experiments.  Freeman  and  Johnson  (1911,  p.  22)  made 
100  inoculations  but  none  was  successful.  They  report,  however,  that 
Derr  was  able  to  make  transfers. 

Inoculations  under  ordinary  conditions — 

The  fact  that  the  oat  rust  is  transferred  with  great  difficulty  to 
wheat  became  apparent  very  soon.  Out  of  108  inoculations  only  one 
was  entirely  successful,  although  5  leaves  became  slightly  flecked. 
Very  evidently  the  infection  threads,  after  having  grown  among  the 
tissues  of  the  leaf,  must  have  died,  since  only  very  minute  flecks  de- 
veloped, and  these  in  only  a  few  cases. 

Effect  of  anesthetics — 

Plants  were  inoculated  after  exposure  to  ether  and  chloroform 
for  periods  of  from  1  to  15  minutes.  The  best  results  were  obtained 
after  exposure  to  ether  for  5  minutes.  In  this  case  3  leaves  out  of  50 
developed  pustules  while  all  the  other  leaves  were  flecked.  These 
pustules  and  flecks  appeared  10  days  after  inoculation,  this  being 
slightly  longer  than  a  normal  incubation  period.  None  of  the  check 
plants  showed  any  distinct  signs  of  successful  infection,  while  the 
flecks  on  the  plants  which  had  been  exposed  to  ether  were  very  con- 
spicuous, forming  a  sharp  contrast  with  the  check  plants.  These 
flecks  were  scattered  all  along  the  line  of  inoculation  and  had  the 
appearance  of  young,  unruptured  pustules. 

This  series  offered  the  best  evidence  that  anesthetics  may  have 
some  influence  in  rendering  a  plant  more  susceptible  to  a  rust  than  it 
would  otherwise  be.  In  subsequent  trials,  exposing  the  plants  for  a 


18  A  STUDY  IN  CEREAL  RUSTS 

greater  length  of  time,  no  pustules  were  developed  on  any  of  the  40 
leaves  inoculated.  Nine  of  these  leaves  were,  however,  quite  strongly 
flecked.  The  use  of  chloroform  did  not  result  in  the  formation  of 
pustules,  although  there  was  a  distinct  advantage  over  the  check  plants 
both  in  number  and  definiteness  of  flecks.  In  all,  40  plants  were  inocu- 
lated and  of  these  19  became  distinctly  flecked.  After  the  use  of  ether, 
then,  3  out  of  90  leaves  were  successfully  infected,  and  56  were  sharply 
flecked,  and  these,  added  to  the  totals  after  the  use  of  chloroform,  give 
3  pustules  and  75  infected  leaves  which  developed  no  pustules  out  of 
130  trials. 

Effect  of  manure — 

Wheat  was  planted  in  two  pots  containing  a  rich  loam  very  heavily 
fertilized  with  rich  barnyard  manure.  Of  the  20  leaves  inoculated,  6 
developed  pustules  and  the  rest  were  strongly  flecked.  In  this  case, 
however,  there  was  a  possibility  that  the  pustules  developed  as  a  result 
of  accidental  infection.  The  flecks  were  apparently  due  to  the  arti- 
ficial inoculation,  since  such  flecks  have  never  been  observed  after 
direct  inoculation  with  spores  from  either  wheat  or  barley,  or  after 
the  inoculation  of  wheat  with  wheat  or  barley  rust.  It  is  therefore 
quite  certain  that  the  pustles,  which  were  normal,  were  the  result  of 
accidental  infection,  while  the  flecks,  which  were  exactly  like  those 
developed  on  a  semi-immune  form,  were  the  result  of  artificial  inocula- 
tion. 

Effect  of  leaf  injury — 

In  one  experiment,  16  leaves  were  pricked  full  of  holes  in  an 
area  of  one  centimeter  or  more.  They  were  then  inoculated,  and  4 
became  flecked,  but  no  pustules  developed.  In  another  experiment  the 
epiderm  was  stripped  from  29  leaves  immediately  before  inoculation. 
Although  10  became  flecked,  the  flecks  were  extremely  minute  and  no 
pustules  were  developed.  Histological  examination  showed  that  the 
spores  had  sent  out  germ  tubes  in  large  numbers.  These  tubes  grew 
among  the  host  cells,  but  true  infection  did  not  take  place.  Sections 
of  these  plants  were  made  and  examined.  It  was  clearly  evident  that 
leaf  injury  did  not  increase  the  chances  for  infection.  The  hyphae  did 
not  develop  better  than  did  those  in  normally  inoculated  plants. 

Summary  of  inoculations  on  wheat — 

Out  of  a  total  number  of  283  leaves  inoculated  under  varying 
conditions  only  4  developed  pustules  and  113  became  flecked,  showing 
that,  although  the  rust  of  oats  can  sometimes  develop  on  wheat,  it  can 
seldom  attain  to  pustule  formation.  The  severity  of  infection,  always 
very  slight,  can  be  increased  somewhat  by  exposing  plants  to  be  in- 
oculated to  anesthetics.  The  experiment  with  high  fertilization  of 


BIOLOGIC  FORMS  19 

soil  seems  to  indicate  that  here,  as  well  as  in  the  case  of  rye,  inoculated 
with  oat  rust,  the  mycelium  is  enabled  to  develop  more  extensively 
and  possibly  produce  more  spores  than  when  ordinary  soil  is  used. 

INOCULATIONS  ON   BARLEY 

The  results  of  this  series  of  inoculations  were  very  similiar  to 
those  obtained  by  other  investigators.  The  behavior  of  the  rust  was 
typical  of  its  usual  behavior  on  an  uncongenial  host  plant.  The  flecks 
were  small  in  nearly  all  cases  and  were  especially  sharp  when  plants 
had  been  exposed  to  anesthetics. 

Inoculations  made  under  ordinary  conditions — 

These  were  not  especially  successful.  No  pustules  were  developed 
on  50  inoculated  leaves  but  10  of  the  leaves  became  flecked. 

Inoculations  after  exposure  to  anesthetics — 

Ether  and  chloroform  were  used.  The  period  of  exposure  varied 
at  different  times  from  5  to  15  minutes.  Plants  exposed  only  5  minutes 
did  not  appear  different  from  the  check  plants.  After  exposure  for 
15  minutes  the  flecking  was  more  distinct  than  on  the  check  plants. 
Only  one  pustule  developed  in  the  entire  number  of  trials,  this  being 
after  exposure  to  ether.  There  were  47  leaves  inoculated  after  expo- 
sure to  ether,  and  of  these  one  developed  a  pustule  and  15  were  fleck- 
ed. The  flecks  in  this  case  had  every  appearance  of  unruptured  pus- 
tules. No  pustules  were  developed  after  exposure  to  chloroform, 
but  7  leaves  out  of  50  became  flecked.  Of  147  leaves  inoculated  32 
were  flecked,  but  only  one  showed  a  pustule. 

SUMMARY  OF  INOCULATIONS  MADE  WITH  PUCCINIA  GRAMINIS  AVENAE 
Oat  rust  to: 

Wheat  -j-J-g- ;  5  flecked 

Wheat  after  exposure  to  ether  -£•$ ;  56  flecked 

Wheat  after  exposure  to  chloroform  -^  ;  19  flecked 

Wheat  after  leaf  injury^;  14  flecked 

Wheat  plants  grown  in  manured  soil  -^V  ? 

Barley^;  10  flecked 

Barley  after  exposure  to  ether  -^  ;  15  flecked 

Barley  after  exposure  to  chloroform  ^^  j  7  flecked 

Rye  -j^-  13  flecked 

Rye  after  exposure  to  ether  -^-f;  26  flecked 

Rye  after  exposure  to  chloroform  ^;  13  flecked 

Rye  after  exposure  to  nitrous  oxide  -f^;  6  flecked 

Rye  after  leaf  injury  ^;  14  flecked 

Rye  grown  in  highly  fertilized  soil  - ;  9  flecked 


20  A  STUDY  IN  CEREAL  RUSTS 

The  rust  can  be  transferred  successfully  from  oats  to  any  one  of 
the  other  cereals.  It  infects  rye  much  more  easily  than  wheat  or  bar- 
ley. Anesthetics  help  to  break  down  the  barriers  as  does  a  high  degree 
of  soil  fertilization.  It  should  be  mentioned  that  this  fact  seems  due, 
not  to  any  new  ability  the  rust  fungus  has  of  attacking  an  uncongenial 
host,  but  to  an  increased  capacity  for  development.  Here  again  evi- 
dence of  the  possible  nature  of  resistance  is  offered  by  the  inoculated 
plants.  The  flecks,  whether  large  or  small,  consist  in  many  cases  of 
dead  tissue  of  the  host  plant.  Histological  examination  shows  that  the 
fungus  gains  entrance  but  cannot  develop  to  any  extent  in  these  areas 
which  are  killed  by  the  fungus  itself. 

EXPERIMENTS  WITH  PUCCINIA  GRAMINIS  SECALIS 
These  experiments  were  not  very  extensive  and  were  made  with 
the  idea  of  determining  rather  the  character  than  the  number  of  suc- 
cessful infections. 

INOCULATIONS  ON  WHEAT 

In  all  70  leaves  were  inoculated.  None  of  them  produced  pustules, 
but  18  became  flecked.  The  flecks  were  distinct  but  in  no  case  were 
they  large.  There  seemed  to  be  much  less  dead  tissue  than  is  the  case 
in  many  of  the  other  forms,  as  for  instance  rye  inoculated  with  barley 
rust.  In  fact  the  flecks  were  hardly  noticeable  unless  one  looked  very 
carefully  for  them.  Apparently  they  were  due  to  the  death  of  host- 
plant  cells  in  the  inoculated  area. 

INOCULATIONS  ON  OATS 

In  addition  to  a  small  number  of  control  inoculations,  some  were 
made  after  exposure  to  ether  and  others  after  exposure  to  chloroform. 
The  anesthetics  apparently  made  no  particular  difference  in  the  suc- 
cess of  the  attempts,  although  there  were  more  flecked  leaves  after  ex- 
posure to  chloroform.  Nothing  out  of  the  ordinary  appeared  so  only 
the  summaries  are  given : 

After  chloroform,  5  min.  T^-;  5  slightly  flecked 
After  ether,  5  min.  -^ ;  2  slightly  flecked 
Under  ordinary  conditions  -^;  1  slightly  flecked 

INOCULATIONS   ON    BARLEY 

After  exposure  to  ether  for  5  minutes,  16  out  of  21  inoculated, 
leaves  became  very  distinctly  flecked.  There  were  many  of  these 
flecks  all  along  the  line  of  inoculation.  They  were  very  suggestive  of 
the  flecking  in  other  forms  in  which  the  mycelium  was  known  to  have 
spread  to  some  extent.  However,  no  pustules  were  developed.  Judg- 
ing from  the  experience  with  other  forms,  if  these  plants  had  been 
highly  fertilized  they  might  have  developed  pustules.  Of  the  check 


BIOLOGIC  FORMS  21 

plants,  only  4  out  of  40  became  flecked.    No  pustules  were  developed. 

INOCULATIONS  ON  EINKORN 

Einkorn  was  inoculated  with  rye  rust  both  after  exposure  to 
ether  for  15  minutes  and  without  having  been  so  exposed.  The  length 
of  time  in  ether  fumes  was  15  minutes.  No  pustules  developed  on 
any  of  the  leaves,  20  being  used  in-  each  series. 

EXPERIMENTS  WITH  PUCCINIA  GRAMINIS  TRITICI 

INOCULATIONS   ON   BARLEY 

It  has  long  been  a  well-established  fact  that  stem  rust  from  wheat 
can  easily  attack  barley.  The  infection  in  trials  made  by  the  writer 
was  found  to  be  normal  and  practically  as  virulent  as  if  spores  had 
been  taken  directly  from  barley.  In  the  one  series  tried  especially  for 
the  purpose  30  out  of  30  leaves  became  characteristically  infected. 
There  is  no  evidence  whatever  of  uncongeniality  between  host  and 
parasite. 

INOCULATIONS  ON  RYE 

Inoculation  under  ordinary  conditions — 

In  the  control  experiments  6  out  of  30  leaves  developed  pustules 
and  20  were  strongly  flecked.  In  fact,  they  were  so  strongly  flecked 
that  flecking  hardly  expresses  properly  the  appearance  developed. 
Long  areas  on  the  leaf  were  killed  outright.  In  these  dead  areas  very 
small  green  islands  were  often  found,  some  of  which  contained  un- 
ruptured  pustules.  All  the  pustules  were  very  minute,  and  some  had 
ruptured  the  epiderm.  It  was  another  very  good  example  of  semi- 
compatibility  between  host  and  fungus.  The  host  leaves  did.  not  suffer 
very  great  injury;  the  fungus  was  enabled  to  spread  to  a  certain  ex- 
tent but  succeeded  in  producing  only  small  pustules. 

Inoculation  after  exposure  to  ether  for  five  minutes — 

The  results  here  were  very  striking.  Fifty  leaves  were  inoculated, 
6  of  which  produced  pustules,  while  every  leaf  was  infected  with  the 
mycelium  of  the  rust  fungus.  The  character  of  the  infection  was  much 
the  same  as  was  that  on  the  check  plants.  The  areas  were  perhaps 
more  extended.  On  some  leaves  there  were  dead  areas  3  centimeters 
long,  while  ordinarily  they  were  not  so  long  on  the  check  plants. 

INOCULATIONS  ON  OATS 

Inoculations  under  ordinary  conditions — 

Direct  inoculation  of  oats  by  spores  from  wheat  has  not  met  with 
success  on  the  part  of  either  Carleton  (1899,  p.  54),  who,  however, 
reports  a  doubtful  case,  or  Freeman  and  Johnson  (1911,  p.  18),  who 
cite  Derr  as  authority  for  the  statement  that  this  direct  transfer  can 
be  made. 


22  A  STUDY  IN  CEREAL  RUSTS 

Attempts  to  transfer  directly  from  wheat  were  unsuccessful.  Not 
a  large  numbe/  were  made,  but  of  the  46  attempts  no  leaves  produced 
pustules  and  only  2  of  them  developed  flecks.  The  flecks  were  very 
small  and  not  very  distinct.  In  this  connection  it  may  be  mentioned 
that  when  oat  leaves  were  inoculated  with  aecidiospores  derived  from 
wheat  teleutospores  one  leaf  out  of  56  developed  pustules.  This  ap- 
pears to  indicate  that  direct  transfer  can  be  made.  The  success  of  the 
inoculation  was  probably  not  due  to  the  fact  that  the  rust  had  passed 
through  the  barberry  stage.  This  seems  especially  true  since  the 
other  cereals  behaved  in  the  same  way  toward  the  aecidiospores  as 
they  did  toward  the  corresponding  uredospores. 

Inoculation  after  exposure  to  ether- 
Only  30  leaves  were  inoculated  after  having  been  in  ether  fumes 
for  from  3  to  5  minutes.  No  pustules  were  developed,  but  10  of  the 
leaves  were  flecked.  There  was  a  perceptible  difference  between  the 
check  plants  and  those  of  this  series,  although  it  cannot  be  said  that 
the  results  were  very  striking. 

INOCULATIONS  ON  OATS  AND  RYE  AFTER  THE  USE  OF  BARLEY  AS  A 

BRIDGING  FORM 

The  work  done  on  bridging  species  has  already  been  mentioned. 
Since  it  is  almost  impossible  to  infect  oats  directly  with  wheat  aecidio- 
spores or  uredospores,  attempt  was  made  to  transfer  aecidiospores  de- 
veloped from  wheat  teleutospores  first  to  barley  and  then  to  infect 
oats  with  the  resulting  uredospores.  The  attempt  was  made  with 
fourth-  and  fifth-generation  uredospores  from  barley,  these  uredo- 
spores having  been  derived  from  wheat-rust  aecidiospores.  Successful 
infection  took  place  in  one  out  of  39  attempts,  4  leaves  being  distinctly 
flecked. 

The  rye  plants  are  possibly  slightly  more  severely  attacked  if  the 
rust  is  transferred  first  to  barley,  16  out  of  19  of  the  plants  inoculated 
becoming  infected. 

SUMMARY  OF  EXPERIMENTS  WITH  PUCCINIA  GRAMINIS  TRITICI 
Wheat  rust  to: 

Barley  fg- 

Rye  -g^;  20  flecked 

Rye  after  exposure  to  ether  -£$;  43  flecked 

Rye  after  barberry  and  4  generations  on  barley  -J-| 

Oats,  uredospores  T°-g-;  2  flecked 

Oats,  aecidiospores    -^ 

Oats  after  exposure  to  ether  -£-$ ;  10  flecked 

Oats  after  barberry  and  4  generations  on  barley  -^ ;  4  flecked 


BIOLOGIC  FORMS 


23 


EFFECT  OF  THE  AECIDIAL  STAGE  ON  BIOLOGIC  FORMS 

GENERAL  STATEMENT 

Eriksson  (1894,  pp.  292-309)  was  one  of  the  first  to  suggest  the 
possibility  of  breaking  down  biologic  forms  by  means  of  the  aecidial 
generation.  He  came  to  the  conclusion,  however,  that  this  could  not 
be  done,  that,  as  far  as  the  cereal  rusts  are  concerned,  they  behave  in 
exactly  the  same  manner  when  transferred  through  barberry  as  they 
do  in  the  urediuial  stage.  Salmon  (1903,  p.  159)  and  Marchal  (1903, 
p.  280)  showed  that  with  biologic  forms  of  the  Erysiphaceae  the  rela- 
tions were  exactly  the  same  whether  ascospores  or  conidia  were  used. 
There  is  not  an  exact  parallelism  between  the  two  since  the  cereal 
rusts  are  heteroecious.  However,  the  cases  are  somewhat  similar  since 
a  sexual  fusion  intervenes  in  both.  Arthur  (1910,  pp.  227-228)  con- 
cludes that  although  there  is  distinct  specialization  of  parasitism  in 
Puccinia  poculiformis  (Jacq.)  Wettst.  (Puccinia  graminis  Pers.)  on 
various  grasses,  this  specialization  breaks  down  in  the  aecidial  stage. 
The  barberry  would,  then,  serve  as  a  bridging  form  between  the  vari- 
ous grasses.  Jaczewski  (1910,  pp.  356-357),  on  the  other  hand,  does 
not  find  this  to  be  the  case  with  biologic  forms  of  Puccinia  graminis 
on  cereals  and  grasses  in  Russia.  His  experiments  support  Eriksson's 
claim  that  the  barberry  does  not  change  the  physiological  specialization 
of  the  various  strains  when  they  produce  aecidia. 

EXPERIMENTS  IN    1912 

In  connection  with  the  experiments  described  by  the  writer  it 
should  be  mentioned  that  the  aecidia  used  in  one  set  of  experiments 
were  developed  in  the  field  but  there  was  very  slight  chance  for  acci- 
dental infection.  Wheat  straw  very  badly  affected  with  rust  in  the 
teleutospore  stage  was  tied  around  barberry  bushes.  There  was  no 
other  rusted  straw  of  any  kind  within  considerable  distance,  so  that 
there  was  little  chance  of  accidental  infection.  These  aecidiospores 
which  were  developed  from  wheat  rust  were  transferred  to  wheat, 
barley,  oats,  rye,  and  einkorn.  The  following  were  the  results : 

RE&ULTS  OF  INOCULATIONS  OF  CEREALS  WITH  AECIDIOSPORES  OF  PUCCINIA  GRAMINIS 
TRITICI  DEVELOPED  IN  THE  FIELD 


Grain 

TRIALS 

Total 

I 

II 

Wheat 

2  1 
TT 

2  9 

"sir 

5  0 
TT 

Barley 

1  2 

T2T 

2  3 
•SIT 

W 

Oats 

A 

1* 
•317 

TT 

Rye 

4 
"2~T 

1  1 
TTB" 

1  5 
-5T 

Einkorn 

1  8 
"317 

24 
"317 

42 
"&17 

•Somewhat  doubtful. 


24 


A  STUDY  IN  CEREAL  RUSTS 


It  will  be  noticed  that  the  percentages  of  infection  are  practically 
the  same  as  are  those  developed  from  uredospore  inoculation.  This 
is  true  also  of  the  character  of  the  infection. 

On  the  rye  plants,  for  instance,  there  was  the  same  characteristic 
spotting  and  the  same  small,  abortive  pustules.  The  pustules  on  rye 
were  all  very  small  and  there  was  no  observable  increase  in  virulence. 
The  same  .thing  is  true  of  einkorn.  On  oats  but  one  rather  doubtful 
pustule  developed,  indicating  that  the  aecidial  stage  in  no  way  broke 
down  the  barriers  in  this  case. 

EXPERIMENTS  IN   1913 

Iii  the  fall  of  1912  the  barberry  bushes  in  the  rust  plat  were  sur- 
rounded with  badly  rusted  wheat  straw,  the  rust  being  in  the  teleuto- 
spore  stage.  In  the  spring  of  1913  the  aecidiospores  were  used  in 
inoculating  the  four  common  cereals  and  einkorn.  The  results  were 
surprising  since  it  was  supposed  that  the  aecidia  had  been  developed 
from  the  wheat-rust  teleutospore  sporidia.  The  results  of  the  various 
trials  are  given  below : 

RESULTS  OF  INOCULATIONS  OF  CEREALS  WITH  AECIDIOSPORES  FROM  FIELD  BARBERRIES 


Grain 

TRIALS 

Total 

I 

II 

III 

Wheat 

8 
TIT 

2 
TT5 

3 

T  U 

1  3 
¥"5" 

Oats 

0 
~fU 

0 
TO" 

0 
~5~Q 

0 

Toir 

Barley 

3  2 

TD" 

1  6 

TT 

1  8 
•3TJ 

6  6 

¥T 

Rye 

40 
TU 

TF 

8 
TT 

5  5 
¥TF 

Einkorn 

1  2 
TTT 

2 

7 

.... 

1  4 
TT 

It  will  readily  be  seen  that  there  would  naturally  be  much  doubt 
as  to  the  origin  of  these  aecidia  from  wheat-rust  teleutospore  sporidia. 
These  aecidiospores  were  being  used  in  spraying  wheats  in  the  rust 
nursery  for  the  purpose  of  developing  a  rust  epidemic.  The  results 
were  very  discouraging.  There  was  a  great  deal  of  Agropyron  repens, 
badly  affected  with  the  teleutospore  stage  of  Puccinia  graminis,  near 
the  barberry  bushes.  Experiments  were  therefore  started  to  determine 
whether  or  not  the  aecidia  on  barberries  might  not  have  been  devel- 
oped from  this  source  rather  than  from  the  wheat-rust  material.  This 
view  seemed  all  the  more  reasonable  when  it  was  observed  that  the 
Agropyron  repens  was  very  severely  affected  with  the  uredospore 
stage  of  rust,  while  the  wheat,  although  it  had  been  thoroughly  and 
persistently  sprayed  with  water  containing  the  aecidiospores,  had  only 
a  few  scattered  pustules  of  rust. 


BIOLOGIC  FORMS 


25 


Barberry  bushes  which  had  been  in  the  cellar  all  winter  were  set 
out  in  the  field  and  covered  with  a  heavy  muslin  cage.  Badly  rusted 
straw  of  wheat  was  tied  around  one  bush  and  that  of  Agropyron  repens 
around  another.  None  of  the  check  plants  developed  any  aecidia  while 
those  surrounded  with  straw  were  very  badly  affected.  The  Agropyron 
repens  material  produced  mature  aecidia  10  days  earlier  than  the  wheat 
material  and  the  aecidia  were  also  developed  in  greater  abundance. 

RESULTS  OF  INOCULATIONS  WITH  AECIDIOSPORES  AND  UREDOSPORES  FROM  WHEAT 

AND  AGROPYRON  REPENS 


Grain 

A.  repens 
aecidia 

A.  repenF 
uredospores 

Wheat 
aecidia 

Wheat 
uredospores 

Wheat 

6 
TFT 

3 

TT 

2  5 

~3~¥ 

30 

~S"0~ 

Oats 

0 
"2T 

0 
~3~TT 

A 

0 

TT 

Barley 

1  2 

-sir 

2  7 
TT 

24 
3TT 

30 
W 

Rye 

1  2 
TT 

2  7 
•STF 

7 

TH 

6 

•anj 

The  field  barberries  were  very  probably  infected  with  the  rust  from 
Agropyron  repens,  as  will  be  readily  observed  by  referring  to  the  two 
tables.  Further,  the  various  biologic  forms  do  not  show  any  apparent 
change  as  a  result  of  having  been  transferred  to  barberry,  thus  con- 
firming the  results  obtained  in  previous  experiments. 

These  inoculations,  although  not  extensive,  show  quite  clearly 
that  Puccinia  graminis  tritici  and  Puccinia  graminis  from  Agropyron 
repens  do  not  seem  to  develop  any  greater  range  of  infection  possi- 
bility for  cereals  after  having  lived  for  a  time  on  the  alternate  host — 
the  barberry.  The  incubation  period,  even  on  wheat,  barley,  and  ein- 
korn,  was  a  little  longer  in  these  experiments  than  that  of  uredo-devel- 
oped  mycelium. 

ADAPTATION  OF  BIOLOGIC  FORMS  TO  NEW  HOSTS 
Magnus  (1894,  p.  362)  was  one  of  the  first  to  suggest  that  a 
particular  biologic  form  might,  by  constant  association  with  one  host, 
change  its  physiological  capabilities  to  such  an  extent  as  to  make  a 
new  race  out  of  it.  This  view  was  also  expressed  by  Dietel  ( 1899,  pp. 
81  and  113)  who  gave  it  as  his  opinion  that  a  given  rust  formerly  at- 
tacked a  number  of  plants  but  by  long  association  with  one  form  be- 
came narrowed  to  this  form  more  closely,  possibly  retaining  also  a 
somewhat  weakened  capability  of  attacking  other  forms.  These 
authors  distinguish  between  adaptation  races  (Gewohnheitsrassen)  and 
true  biologic  forms,  the  tendency  being,  under  favorable  conditions, 


26  A  STUDY  IN  CEREAL  RUSTS 

for  the  former  to  develop  into  the  latter.  Eriksson  ( 1902,  p.  657)  also 
expresses  this  view  in  a  somewhat  modified  form.  Ward  (1902-1) 
shows  that  adaptation  of  Puccinia  dispersa  takes  place.  Klebahn  ( 1904, 
pp.  152-167)  cites  numerous  experiments  to  show  that  this  may  be 
the  case.  Miss  Gibson  (1904,  pp.  184-191)  grew  a  number  of  suc- 
cessive generations  of  rust  on  resistant  host  varieties,  but,  in  that  time, 
found  little  adaptational  tendency.  Massee  (1904,  p.  17)  explains  the 
resistance  and  susceptibility  of  various  plants  to  parasitic  fungi  on  the 
ground  of  the  presence  or  absence  of  chemotactic  substances  in  the 
host.  He  contends  that  saprophytes  can  be  educated  to  become  para- 
sites. This  would  also  apply  in  large  measure  to  biologic  forms.  Sal- 
mon (1905,  p.  183)  grew  Erysiphe  graminis  from  wheat  on  Hordeum 
sylvaticum  for  five  generations  and  found  no  diminution  in  the  power 
to  infect  the  original  host.  Freeman  and  Johnson  (1911,  p.  28)  con- 
clude that  "the  host  plants  exercise  a  strong  influence,  not  only  on 
the  physiological  and  biological  relationships,  but  in  some  cases  even 
on  the  morphology  of  the  host." 

An  attempt  was  made  to  test  this  matter.  The  object  was  to  de- 
termine the  change,  if  any,  in  the  physiology  of  the  rust  as  evidenced 
by  its  power  of  infection  and  in  its  morphology  as  evidenced  "by  changes 
in  spore  dimensions.  For  this  purpose  Puccinia  graminis  tritici  was 
grown  on  Minnesota  No.  163,  a  susceptible  wheat,  and  on  einkorn  2433. 
The  einkorn,  in  the  first  few  trials,  was  apparently  one  of  the  most 
resistant  of  the  Triticums  to  the  wheat  stem  rust.  The  rust  was  trans- 
ferred to  einkorn  and  grown  on  this  host  through  successive  genera- 
tions for  19  months,  transfers  being  made,  on  an  average,  once  every  3 
weeks.  After  one  year  on  einkorn  the  rust  seemed  to  be  much  more 
virulent  than  the  original  wheat  rust  had  been.  Einkorn  plants  were 
inoculated  with  this  einkorn  rust  and  with  uredospores  from  wheat. 
In  both  cases  there  was  100  per  cent  of  infection,  but  the  virulence  of 
the  infection  was  quite  different.  On  the  leaves  inoculated  with  rust 
from  einkorn,  leaf  areas  one  to  two  centimeters  long  were  affected, 
being  well  covered  with  pustules  shedding  spores  in  great  abundance. 
Individual  pustules  were  from  one  to  three  millimeters  long  and  nearly 
as  broad,  giving  every  indication  of  a  severe  rust  attack.  On  the  plants 
inoculated  with  wheat  rust,  on  the  other  hand,  the  pustules  were  al- 
ways smaller,  although  fairly  numerous.  Few  of  them  were  as  much 
as  one  millimeter  long  and  many  of  them  did  not  rupture  at  all. 

Some  wheat  plants  were  inoculated  with  rust  from  einkorn  and 
others  with  the  rust  taken  directly  from  wheat.  Here  again  there  was  a 
considerable  difference  in  virulence  of  infection.  The  pustules  developed 
from  einkorn-rust  inoculations  were  fairly  large  and  numerous.  Some 
of  them  were  two  millimeters  long,  although  the  average  length  was 
less  than  this.  The  infection  on  wheat  inoculated  with  wheat  rust  was 


BIOLOGIC  FORMS  27 

much  more  severe.  Areas  of  the  leaf  two  centimeters  in  length  were 
often  almost  covered  with  pustules,  some  of  which  attained  a  length  of 
seven  millimeters. 

A  number  of  other  trials  were  made,  the  last  after  the  rust  had 
been  confined  to  einkorn  for  17  months.  In  one  experiment  the  re- 
sults were  just  the  opposite  of  what  were  expected.  This  is  explained 
by  the  fact  that  lack  of  greenhouse  space  necessitated  keeping  the  ein- 
korn plants  inoculated  with  einkorn  rust  in  a  draught  of  cold  air. 
There  were  only  a  few  wheat  uredospores  available  in  inoculating  the 
wheat,  so,  in  this  experiment,  einkorn-by-einkorn  inoculations  were  not 
so  successful  as  einkorn-by-wheat.  In  subsequent  trials,  however,  un- 
der uniform  conditions,  the  results  first  described  were  substantiated. 

The  conclusion,  then,  is  justified  that  by  confining  Puccinia  gram- 
mis  tritici  to  einkorn  for  successive  generations  throughout  a  year  or 
more  the  rust  adapts  itself  somewhat  to  its  new  host  and  loses,  at  least 
to  a  slight  degree,  its  power  to  infect  the  original  host.  It  would  no 
doubt  require  a  very  long  period  of  time  to  fix  this  character  in  the 
plant  to  such  a  degree  as  to  make  it  a  new  biologic  form.  But  there  is 
very  evidently  such  an  adaptational  tendency  (see  Plates  II  and  III). 
It  must  be  noted,  however,  that  this  new  character  is  not  so  firmly 
fixed  that  it  cannot  be  overbalanced  by  environmental  factors.  The  ex- 
perimental production  of  new  forms  is  apparently  possible,  but  a  long 
period  of  time  is  required. 

The  change  in  the  fungus  manifests  itself  not  merely  in  the  para- 
sitic tendency  toward  the  host  but  in  the  morphology  as  well.  Wheat 
and  einkorn  were  inoculated  with  spores  from  the  same  plant.  The 
uredospores  after  growing  for  a  year  on  wheat  averaged  37.85x22.76 /* 
while  those  grown  on  einkorn  for  a  year  measured  33.58x21.79  /*. 
When  einkorn  was  inoculated  with  Puccinia  graminis  tritici  aecidio- 
spores,  the  resulting  uredospores  were  more  nearly  identical  with  the 
wheat-rust  spores  in  length.  The  width,  however,  remained  practically 
the  same.  The  average  size  of  these  spores  was  35.92x21.69  /*. 

SUMMARY  OF  PART  I 

1.  Direct  transfers  of  Puccinia  graminis  have  been  made  from 
oats  to  both  wheat  and  rye.    The  rusts  from  oats  and  barley  used  in 
these  experiments  could  be  transferred  to  rye  more  easily  than  those 
used  by  Carleton  or  those  used  by  Freeman  and  Johnson.    The  barley 
rust,  however,  did  not  prove  as  versatile  as  the  strain  used  by  Freeman 
and  Johnson. 

2.  The  use  of  anesthetics  has  some  effect  in  rendering  an  immune 
plant  slightly  more  susceptible  to  the  rust;  leaf  injury  apparently  had 
no  effect. 

3.  High  fertilization,  by  increasing  the  virulence  of  the  attack 


28  A  STUDY  IN  CEREAL  RUSTS 

on  semi-immune  forms,  may  have  some  influence  in  breaking  down 
biologic  forms. 

4.  There  appears  to  be  a  physiological  and  even  a  slight  mor- 
phological change  in  the  rust  fungus  when  grown  continuously  on  a 
semi-immune  host.  The  physiological  change  manifests  itself  as  an 
adaptation  to  the  new  host,  which,  however,  is  very  gradual. 

i  5.  There  are  indications  that  biologic  forms  of  cereal  rusts,  at 
least  Puccinia  graminis  tritici,  do  not  lose  their  specialization  tenden- 
cies when  grown  on  barberry. 

6.  The  degree  of  incompatibility  of  host  and  parasite  varies 
greatly.  In  semi-compatible  forms,  fairly  large  leaf  areas  are  some- 
times killed,  indicating  a  killing  of  host  cells  by  fungus  and  consequent 
death  of  the  mycelium  itself.  In  this  respect  they  resemble  very  closely 
some  of  the  rust  resistant  forms  of  wheat.  The  biologic  forms  of  rusts, 
therefore,  with  susceptible  and  immune  varieties  of  host  plants,  throw 
light  on  the  question  of  the  nature  of  resistance  to  Puccinia  graminis. 


PART  II.     RUST-RESISTANT  VARIETIES  OF  WHEAT 

HISTORICAL 

It  has  long  been  a  matter  of  common  observation  that  some  wheats 
are  more  resistant  than  others  to  the  attacks  of  Puccinia  graminis  and 
other  rusts.  Among  the  earlier  observers  Henslow  (1841,  p.  3),  La 
Cour  (1863,  p.  326)  and  Little  (1883,  p.  634)  note  that  some  wheats 
are  less  injured  by  rust  than  are  others.  Bolley  (1889,  p.  16)  observes 
that  those  varieties  least  susceptible  to  rust  are  "hardy,  stiff-strawed 
wheats,  having  smooth,  fibrous  leaves."  Anderson  (1890,  p.  84)  says 
that  hard,  flinty  wheats  are  more  rust-resistant  than  others.  He  thinks 
it  may  be  due  to  the  large  amount  of  silica  in  the  hard  wheats.  Cobb 
(1892)  advanced  his  "mechanical  theory"  to  explain  resistance.  It 
was  due,  according  to  his  idea,  to  morphological  characters  of  the 
host,  namely,  thick  cuticle,  waxy  coating,  and  small  stomata.  Hitch- 
cock and  Carleton  (1893,  p.  12)  also  correlated  resistance  with  morpho- 
logical characters,  asserting  that  resistant  forms  had  stiff,  upright 
leaves  with  a  thick  epidermis.  Eriksson  (1895,  p.  199)  and  many 
others  since  have  shown,  however,  that  a  wheat  resistant  to  one  spe- 
cies of  rust  is  not  necessarily  resistant  to  another  species,  thus  indicat- 
ing a  rather  delicate  relationship  as  the  basis  of  resistance. 

Eriksson  and  Henning  (1896,  pp.  332-365)  were  unable  to  sub- 
stantiate Cobb's  mechanical  theory,  since  morphological  characters 


RUST-RESISTANT  VARIETIES  OF  WHEAT  29 

could  not  always  be  correlated  with  resistance.  They  suggest  that  re- 
sistance is  of  a  complex  chemico-physiological  nature  and  is  inherent 
and  fairly  constant  within  the  plant.  Ward  (1902)  decided  in  connec- 
tion with  Puccinia  dispersa  on  the  bromes  that  resistance  was  indepen- 
dent of  any  recognizable  morphological  character  and  suggested  that 
the  problem  was  much  the  same  as  that  dealing  with  the  factors  gov- 
erning fertility  and  sterility  of  stigmas  to  pollen.  Biffen  (1907,  p.  128) 
concludes  that  resistance  to  Puccinia  glumarum  is  independent  of  any 
discernible  morphological  character.  He  reasserts  this  principle  in  a 
later  work  (1912,  pp.  421-429).  Bolley  (1908),  although  not  positive, 
inclines  to  the  view  that  disease-resistance  is  physiological  rather  than 
morphological  in  its  nature.  Cook  and  Taubenhaus  (1912)  show  that 
various  vegetable  acids  are  toxic  to  parasitic  fungi  and  that  the  amount 
of  some  of  these  acids  present  depends  on  the  stage  of  ripening  of  the 
fruit.  Jones,  Giddings,  and  Lutman  (1912,  p.  83)  conclude  that  the 
resistance  of  potato  tubers  and  leaves  to  Phytophthora  infestans  is  due 
to  somtthing  either  largely  or  wholly  within  the  tissues.  The  consen- 
sus of  opinion  among  the  more  recent  investigators  seems  to  be  that 
there  is  a  very  delicate  balance  maintained  between  host  and  parasite. 
This  balance  is  dependent,  to  a  certain  extent,  on  the  environmental 
conditions  under  which  host  plants  are  grown.  This,  however,  as  well 
as  other  phases  of  the  problem,  will  be  discussed  more  fully  under  the 
various  sections  into  which  the  question  naturally  subdivides  itself. 

FORMS  WHICH  ARE  RESISTANT 

Attention  has  frequently  been  called  by  various  observers  to  the 
fact  that  freedom  from  disease  does  not  necessarily  indicate  resistance. 
Varieties  which  mature  early  may  escape  the  disease,  and  various  other 
factors,  mainly  ecological,  may  influence  the  degree  of  resistance. 
Careful  experiments,  however,  have  shown  that  some  varieties  of 
Triticum  durum  are  really  resistant.  The  resistance  of  durum  wheats 
varies  with  locality  according  to  Bolley's  observations  (1906,  p.  662). 
Consequently  to  determine  absolute  resistance  the  plants  should  be 
grown  under  controlled  conditions.  Further,  they  should  be  subjected 
to  conditions  favorable  for  infection. 

FIELD  OBSERVATIONS 

Field  observations  were  made  on  various  forms  which  were  grown 
in  a  rust  plat.  An  epidemic  was  induced  by  spraying  frequently  with 
water  containing  a  large  number  of  spores  of  Puccinia  graminis  tritici. 
Frequent  observations, were  made  on  the  amount  of  rust.  Final  notes 
were  taken  at  ripening  time.  The  percentage  indicates  in  each  case 
the  estimated  percentage  of  resistance,  assuming  absolutely  immune 
forms  to  have  a  resistance  of  100  per  cent. 


30  A  STUDY  IN  CEREAL  RUSTS 

ESTIMATED  PERCENTAGES  OF  RUST  ON  VARIETIES  GROWN  IN  1911  AND  1912 

1911  1912 

Per  Per 

Variety                                       Cent  Variety                                       Cent 
Einkorn  2433 

Dickinson  1910  Ped.  No.  4..  92  Minnesota  No.   169 15 

No.  6  90  lumillo  1736 98 

No.  7  93  Minnesota  No.  188 30 

No.  8  95  Einkorn  2433  96 

lumillo  1736  95  Khapli   95 

Kubanka  1516  No.  8 15  Minnesota  No.  169 35 

Kubanka  1516  No.  9 10  Arnautka    288 97 

Minnesota  No.  163 10  Arnautka  1431  67 

It  will  be  noticed  that  in  1912  the  resistance  was  slightly  greater 
than  in  1911.  This  may  be  accounted  for  by  the  fact  that  in  1911  the 
grains  were  sown  late,  giving  the  rust  ample  opportunity  to  develop 
fully. 


EXPERIMENTAL 

GREENHOUSE  TRIALS 

The  varieties  tested  for  resistance  in  the  greenhouse  were :  Minne- 
sota No.  163,  Minnesota  No.  169,  Kubanka  1516,  Nos.  8  and  9,  Ku- 
banka 2094,  lumillo  1736,  einkorn  2433,  Nos.  4,  6,  7,  and  8,  emmer 
1522,  Arnautka  288,  and  Khapli.  Of  these,  Minnesota  No.  163,  Min- 
nesota No.  169,  Kubanka  1516  did  not  prove  resistant.  The  behavior 
of  einkorn  has  already  been  discussed  under  adaptation  of  biologic 
forms.  Very  careful  observations  were  made  on  the  others  to  deter- 
mine as  accurately  as  possible  their  comparative  resistance.  The  inocu- 
lations were  made  with  fresh,  viable  spores  and  the  plants  put  under 
bell  jars  48  hours  after  inoculation. 

The  incubation  period  varies  with  temperature  conditions,  both 
high  and  low  temperatures  lengthening  the  period  very  perceptibly. 
No  experiments  with  this  particular  object  in  view  were  made,  but 
numerous  observations  brought  the  fact  out  very  clearly.  Under  the 
same  conditions  on  Minnesota  No.  163  the  incubation  period  is  shorter 
than  on  any  of  the  resistant  forms.  With  an  average  temperature  of 
about  65  degrees  Fahrenheit  and  a  variation  of  from  40  to  75  degrees 
and  a  relative  humidity  of  about  55  per  cent,  pustules  appear  on  Minne- 
sota No.  163  in  7  or  8  days.  In  case  of  lumillo  the  period  is  usually 
about  2  days  longer,  although  considerable  variation  was  found.  Em- 
mer has  an  incubation  period  of  about  11  days,  Arnautka,  12  days,  and 
Khapli,  14  days.  Arranged  in  order  of  their  susceptibility  these  vari- 
eties are  as  follows : 


RUST-RESISTANT  VARIETIES  OF  WHEAT  31 


Variety  Incubation  Period 

Minnesota  No.'  163 7  days 

lumillo  1736   9  days 

Emmer  1522  11  days 

Arnautka  288   12  days 

Khapli 14  days 

It  will  thus  be  seen  that  the  incubation  period  is  longer  on  the  more 
resistant  varieties.  These  figures  are,  of  course,  not  absolute,  but  vary 
with  the  temperature,  and,  to  some  extent,  with  soil  conditions.  All 
however,  vary  in  nearly,  but  not  absolutely,  the  same  proportion. 

The  character  of  infection  is  distinctly  different  on  the  different 
varieties.  It  is  quite  noticeable  that  the  same  phenomena  are  observed 
as  appear  on  various  biologic  forms.  On  Minnesota  No.  163  the  pus- 
tules are  large,  varying  from  2  to  6  millimeters  in  length.  They  rup- 
ture the  epiderm  very  readily  and  shed  spores  in  great  abundance. 
Very  rarely  are  small,  unruptured  pustules  developed.  The  host  tissues 
nearly  always  remain  fairly  healthy,  a  yellowing  which  gradually  ap- 
pears furnishing  the  external  evidence  that  the  fungous  hyphae  are  in 
the  plant.  On  einkorn  and  lumillo,  which  in  the  greenhouse  are  only 
fairly  resistant,  the  pustules  are  usually  somewhat  smaller.  There  is 
a  tendency  in  these  two  forms  toward  the  development  of  small  dead 
areas.  These  areas  are  either  very  distinctly  yellowed  or  sometimes 
killed  outright.  The  general  appearance  is,  however,  usually  not 
sharply  different  from  that  of  infected  Minnesota  No.  163,  except  in 
degree  of  infection  as  evidenced  by  smaller  pustules  on  einkorn  and 
lumillo.  On  emmer  there  were  often  long  infected,  yellow  areas  in 
which  there  was  a  fairly  large  number  of  very  small  pustules,  usually 
less  than  one  millimeter  in  length,  many  of  which  never  ruptured. 
Then  again  fairly  large  areas  of  host  tissue  were  practically  killed  and 
only  a  few  small  green  islands  developed.  On  both  Arnautka  and 
Khapli,  areas  from  one  to*two  centimeters  long  were  killed,  the  leaf 
appearing  white  and  dead  (see  Plate  VI,  A).  In  these  areas  there  was 
often  a  moderately  large  number  of  "green  islands"  with  very  small, 
unruptured  pustules  in  the  centers.  When  the  pustules  did  rupture  they 
were  always  very  small,  seldom,  if  ever,  exceeding  one  millimeter  in 
length  and  often  being  mere  dots.  The  large  areas  involved  can  be 
explained  rather  on  the  basis  of  multiple  infection  than  on  the  basis  of 
the  spreading  of  the  mycelium  from  a  few  infections.  Histological 
examination  of  diseased  areas  verifies  this  supposition. 

The  spores  on  the  resistant  varieties  were  smaller  than  on  Minne- 
sota No.  163.  Spores  of  Minnesota  No.  163  averaged  35.38x21.39  /*. 
while  those  of  emmer  were  33.04  x  21.30  //.  The  Khapli  spores  were 
smallest,  being  only  29.69  x  20.68  >w.  It  was  found  that  spores  from 
different  pustules  varied  somewhat  in  average  size.  Therefore  spores 


32  A  STUDY  IN  CEREAL  RUSTS 

from,  a  number  of  pustules  were  measured  in  determining  the  averages. 
The  fact  that  pustules  are  produced  only  with  difficulty  and  that  the 
spores  are  smaller  on  resistant  varieties  would  seem  to  indicate  that 
the  fungus  is  not  vigorous  and  cannot  develop  extensively  although  it 
may  gain  entrance  into  the  leaf  tissues. 

COMPARATIVE     VIRULENCE     OF     AECIDIAL     AND      LONG-TIME 
UREDOSPORE  INOCULATIONS   ON  RESISTANT  FORMS 

Various  observers  have  remarked  on  the  reinvigorating  power  of  the 
aecidial  stage  of  Puccinia  graminis.  Plowright  (1882,  p.  234)  was  of 
the  opinion  that  much  more  damage  was  done  by  aecidial  infections 
than  by  infection  by  uredospores  which  had  been  reproduced  for  a 
number  of  successive  generations.  Bolley  (1889,  p.  13)  states  that  the 
aecidium,  being  a  sexual  product,  should  be  considered  as  functionally 
reinvigorating.  He  also  reasserts  this  principle  in  a  later  work  (1909, 
p.  182).  Arthur  (1902,  pp.  68  and  69  and  1903,  p.  17)  observes  that 
primary  uredospores  have  a  greater  disturbing  effect  on  the  host  than 
do  long-time  uredospores.  Freeman  and  Johnson,  on  the  other  hand, 
cite  experiments  (1911,  p.  33)  to  show  that  when  the  aecidial  and 
teleutospore  stages  were  excluded  for  52  generations  the  fungus  still 
retained  its  power  of  infection.  The  fact  that  sexuality  in  the  rusts  has 
been  definitely  established  would  make  it  seem  reasonable  to  suppose 
that  there  would  be  a  reinvigorating  power.  However,  Barclay  (1892, 
pp.  8  and  40)  states  that  in  India  there  are  no  barberries  for  "enormous 
distances"  from  fields  of  wheat  in  which  Puccinia  graminis  is  quite 
destructive.  McAlpine  (1906,  p.  58)  points  out  that  Puccinia  graminis 
probably  causes  no  more  damage  in  any  country  in  the  world  than  it 
does  in  Australia  where  barberries  are  practically  absent  and  aecidia 
.have  never  been  found.  The  rust  is  quite  serious  in  South  Africa,  but, 
according  to  Pole  Evans  (1911),  the  aecidtal  stage  is  absent. 

Comparative  trials  were  made  with  aecidiospores,  primary  ured- 
ospores, and  long-time. uredospores.  The  varieties  used  were  not  in 
all  cases  the  most  resistant,  since  no  seed  of  some  of  the  more  resist- 
ant forms  was  available  when  the  aecidia  appeared.  Trials  were  made 
on  Minnesota  No.  163,  Kubanka  1516,  lumillo  1736,  and  einkorn  2433. 
The  two  last-mentioned  forms  are  fairly  resistant.  The  long-time  ured- 
ospores used  represented  the  twenty-fourth  generation  on  wheat.  A 
number  of  trials  were  made  and  the  results  were  not  always  uniform. 
The  incubation  period  of  the  fungus  when  developed  from  primary 
uredospore  or  from  aecidiospore  inoculations  was  slightly  longer  than 
when  developed  from  long-time  uredospores.  The  pustules  developed 
from  long-time  uredospores  were  apparently  smaller  and  more  numer- 
ous, while  those  from  aecidiospores  and  primary  uredospores  averaged 


RUST-RESISTANT  VARIETIES  OF  WHEAT  33 

somewhat  larger,  were  deeper  brown,  and  seemed  to  be  shedding 
spores  in  greater  abundance.  On  einkorn,  in  one  experiment,  the  re- 
sults were  directly  opposed  to  this.  The  differences  were  not  especially 
striking,  the  aecidial  infections  being  perhaps  slightly  more  virulent. 
There  is  considerable  evidence  that  the  virulence  of  the  rust  attack 
when  carried  by  aecidiospores  or  primary  uredospores  is  exceptionally 
virulent.  The  results  of  these  experiments,  however,  would  not  justify 
such  a  conclusion  in  this  particular  case. 

METABOLISM  OF  THE  HOST  AND  RUST  RESISTANCE 

There  seems  to  be  no  question  but  that  weather  and  soil  condi- 
tions, determining  the  metabolism  of  the  host  plants,  exert  an  influence 
un  the  prevalence  of  rust  in  the  field.  Little  (1883,  p.  634)  states  that 
weather  is  the  determining  factor  and  adds  that  high  manuring,  espe- 
cially with  nitrogenous  manures,  predisposes  wheat  plants  to  rust. 
Bolley  (1889),  Anderson  (1890),  and  many  others  since  have  held  that 
this  is  the  case.  Bolley  suggests  as  a  possible  cause  the  increased 
evaporation  and  consequent  raising  of  the  relative  humidity.  Jones 
(1905)  shows  that  Phytophthora  rot  of  potatoes  tends  to  be  more  seri- 
ous after  a  heavy  application  of  nitrogenous  fertilizers  to  the  land. 
Miss  Gibson  (1904)  concluded  as  the  result  of  experiments  with  the 
chrysanthemum  rust  that  in  an  almost  immune  form  normal  develop- 
ment of  rust  does  not  depend  on  the  state  of  health  of  the  plant,  but 
that  a  luxuriant  state  of  growth  favors  the  development  of  the  fungus. 
Hennings  (1903,  pp.  41-45),  on  the  other  hand,  states  that  in  observ- 
ing plants  infected  with  perennial  smuts  and  with  rusts  he  found  that 
the  disease  disappeared  when  the  plants  were  placed  under  the  most 
favorable  cultural  conditions.  This  is  not  in  accord  with  Arthur's  gen- 
eralization (1903,  p.  13)  that  "so  intimate  is  the  association  of  host 
and  parasite  that  as  a  rule  the  vigor  of  the  parasite  is  directly  propor- 
tional to  the  vigor  of  the  host."  Apparent  discrepancies  may,  how- 
ever, be  explained  by  the  fact  that  different  plants  and  different  para- 
sites react  quite  differently.  As  far  as  the  rusts  of  cereals  are  con- 
cerned, Arthur's  generalization  would  seem  to  be  correct.  Biffen 
(1912,  pp.  4^21-429)  shows  that  Puccinia  glumarum  is  most  virulent 
when  a  complete  fertilizer  is  used  and  that  the  virulence  decrease? 
with  the  decrease  in  amount  of  fertilizer. 

Less  work  has  been  done  to  determine  the  exact  manner  in  which 
these  causes  operate.  De  Bary  (1887,  p.  359)  says,  "The  physiological 
reason  for  these  predispositions  cannot  in  most  cases  be  exactly  stated ; 
but  it  may  be  said  in  general  terms  to  lie  in  the  material  composition  of 
the  host,  and  therefore  to  be  indirectly  dependent  on  the  nature  of  its 
food.''  Marshall  Ward  (1902,  p.  145),  in  experiments  with  Puccinia 
dispersa  on  bromes,  tried  the  effect  of  mineral  starvation  and  concluded 


34  A  STUDY  IN  CEREAL  RUSTS 

that  "lack  of  minerals  in  no  way  secured  immunity  from  infection, 
although  seedlings  deficient  in  phosphorus  or  nitrogen  tended  to  show 
retardation  of  infection."  The  well-nourished  plants  produced  more 
spores  than  the  underfed  ones.  This  seems  to  be  due  not  so  much  to 
the  presence  or  absence  of  any  particular  chemicals  and  a  direct  effect 
on  the  fungus  but  rather  to  the  effect  on  the  host.  However,  attempts 
have  been  made  to  prevent  diseases,  among  them  rusts  of  cereals,  by 
adding  various  substances  to  the  soil.  Anderson  (1890,  p.  84)  recom- 
mends the  use  of  salt,  iron  sulfate,  and  lime  as  tending  to  decrease  the 
amount  of  rust.  Galloway  (1893,  p.  208)  tried  the  effect  of  flowers  of 
sulfur,  potassium  sulfid,  ammonium  carbonate,  potassium  bichromate, 
calcium  hydroxid,  and  iron  sulfate  when  applied  to  the  soil,  but  found 
them  of  no  particular  value  in  preventing  rust.  Laurent  (1902,  pp. 
1040-1042)  concludes  that  potatoes  can  be  immunized  against  Phytoph- 
thora  infestans  by  treating  the  soil  with  copper  sulfate.  Marchal  (1902, 
pp.  1067  and  1068)  tried  the  effect  of  copper  sulfate  and  iron  sul- 
fate of  various  strengths  when  added  to  Sach's  solution,  on  the  se- 
verity of  attack  of  Bremia  lactucae  on  lettuce.  He  found  that  by  add- 
ing 4  or  5  parts  of  copper  sulfate  to  10,000  parts  of  Sach's  solution 
he  was  able  to  render  the  plants  practically  resistant  to  the  fungus, 
yet  leave  the  vegetation  normal.  In  experiments  attempting  to  immun- 
ize cereals  to  rusts  he  was  unsuccessful.  Massee  (1903,  p.  142)  pre- 
vented the  development  of  fungi  on  tomatoes  and  cucumbers  by  water- 
ing them  with  copper  sulfate  solution.  This  did  not  give  the  desired 
results  with  Oidium  on  barley.  He  states  that  not  all  plants  can  be 
treated  in  this  way  without  endangering  their  health.  Chemical  analy- 
sis of  treated  %and  untreated  tomatoes  showed  that  there  was  no  more 
copper  sulfate  in  treated  and  therefore  immune  plants  than  there  was 
in  those  which  had  received  no  treatment.  Massee  suggests  therefore 
that  the  copper  sulfate  reacts  with  certain  substances  in  the  soil,  thus 
indirectly  conferring  immunity.  Freeman  and  Johnson  (1911,  pp.  69- 
70)  call  attention  to  the  complexity  of  the  problem  and  the  need  for 
differentiating  results. 

EFFECT  OF  WATER  CONTENT  OF  SOIL 

Statements  to  the  effect  that  low-lying,  wet  soils  predispose  cereals 
to  rust  are  frequently  made  in  the  literature  of  the  subject.  Reference 
has  already  been  made  to  some  of  these.  It  was  observed  very  fre- 
quently in  experiments  mentioned  in  this  paper  that  when  the  relative 
humidity  was  high  infection  was  not  only  surer  to  result  but  that  it 
was  also  more  severe.  It  was  therefore  thought  worth  while  to  de- 
termine whether  a  high  water  content  of  the  soil  would  act  as  a  pre- 
disposing factor. 

The  varieties  used  were:    Einkorn  2433,  Kubanka  1516,  lumillo 


RUST-RESISTANT  VARIETIES  OF  WHEAT  35 

1736,  and  Minnesota  No.  163.  There  was  no  difference  in  the  amount 
of  water  until  the  plants  germinated.  Immediately  after  germination, 
however,  the  soil  in  one  series  was  kept  very  wet  while  that  in  another 
series  was  kept  as  dry  as  was  possible  without  endangering  the  life  of 
the  plants.  The  soil  in  the  wet  series  had  a  water  content  of  31.35 
per  cent,  while  that  of  the  dry  series  was  6.16  per  cent  at  the  conclusion 
of  the  experiment.  Repeated  trials  were  made  with  substantially  the 
same  results. 

The  number  of  einkorn  and  lumillo  leaves  which  became  infected 
in  the  wet  series  was  smaller  than  in  the  dry  series.  The  percentages 
for  the  others  were  practically  the  same.  In  virulence  of  infection, 
however,  there  was  considerable  difference.  The  varieties  also  reacted 
somewhat  differently  so  each  will  be  considered  separately. 

On  Kubanka  the  virulence  of  infection,  especially  in  the  early 
stages,  is  very  markedly  inferior  on  the  plants  in  the  wet  series.  The 
pustules  during  the  early  stages  are  often  small  and  on  some  plants 
do  not  appear  at  all,  the  leaf  merely  becoming  yellow.  Later  the  plants 
in  dry  soil  were  often  completely  covered  with  large,  vigorous  pus- 
tules while  those  in  wet  soil,  although  producing  a  moderately  large 
number  of  pustules,  were  not  nearly  so  badly  affected.  In  both  series 
there  were  many  secondary  infections  along  the  leaf.  There  was  a 
distinct  tendency  in  the  wet  series  toward  leaf-yellowing.  It  was  at 
first  thought  that  the  mycelium  might  be  spreading  through  the  tissues. 
Histological  examination,  however,  failed  to  confirm  this  supposition. 
Apparently  it  was  only  a  slight  chlorotic  condition  due  to  excessive 
water  content.  The  infection  was  unquestionably  more  severe  on  plants 
grown  in  dry  soil  (see  Plate  IV). 

On  einkorn  the  differences  were  not  so  sharp,  although  there  ap- 
peared to  be  a  slightly  more  severe  infection  on  plants  in  the  dry  series 
than  on  those  in  the  wet  series.  The  rust  appeared  at  about  the  same 
time,  the  virulence  of  infection  being  at  first  quite  distinctly  greater 
on  the  dry-soil  plants.  Later  this  difference  was  not  quite  so  marked, 
although  still  apparent. 

The  sharpest  difference  was  on  lumillo.  Only  a  few  leaves  in  the 
wet  series  were  really  badly  infected,  while  those  of  the  dry  series 
showed  a  surprisingly  virulent  attack.  There  is  no  question  but  that 
the  infections  secured  on  plants  in  these  dry  series  were  more  severe 
than  were  those  on  any  other  lumillo  plants  inoculated  during  the 
various  trials  with  this  variety.  It  is  not  often  that  a  "really  vigorous 
development  of  the  fungus  occurs  on  lumillo,  but  when  the  water  con- 
tent of  the  soil  is  very  low,  the  infection  at  times  shows  surprising  vir- 
ulence (see  Plate  V). 

The  results  on  Minnesota  No.  163  varied  more  than  those  of  the 
other  forms.  The  pustules  in  nearly  every  case  were  large  and  vigor- 


36  A  STUDY  IN  CEREAL  RUSTS 

ous  with  but  little  difference  between  the  two  series.  There  seemed  to 
be  a  tendency  for  the  mycelium  to  spread  more  in  the  plants  grown  in 
wet  soil,  but  the  pustules  were  not  larger  than  those  on  dry-soil  plants. 
Whatever  difference  there  was  appeared  as  a  slightly  greater  virulence 
on  the  wet-soil  plants. 

It  will  be  noticed  that  lumillo  and  Kubanka,  drought-resisting 
plants,  were  more  severely  attacked  when  grown  in  dry  soil.  Minne- 
sota No.  163,  on  the  other  hand,  a  mesophyte,  did  not  show  so  much 
difference.  It  is  probable  then,  that,  conditions  having  been  favorable 
for  a  rust  infection,  the  water  relation  in  the  soil  which  is  most  favor- 
able for  the  host  plant's  development  is  also  the  most  favorable  for  the 
development  of  the  rust.  It  seems  probable  also  that  in  at  least  some 
forms  it  is  not  the  water  content  of  the  soil  which  predisposes  grains 
growing  in  low  places  to  rust  but  rather  the  increased  relative  humidity 
which  enables  the  rust  spores  to  germinate  and  infect  the  plants.  The 
temperature  in  such  places  also  probably  exerts  an  influence.  This  is 
pointed  out  by  Freeman  and  Johnson  (1911,  p.  65)  in  connection  with 
the  rust  epidemic  of  1904. 

EFFECT  OF  FERTILIZERS 

In  the  first  series  the  varieties  used  were :  Minnesota  No.  163, 
Arnautka  288,  Khapli,  emmer  1522,  lumillo  1736,  einkorn  2433,  and 
Kubanka  2094.  Some  were  planted  in  ordinary  rich  loam,  others  in 
rich  loam  plus  fresh  barnyard  manure,  and  a  third  series  in  rich  loam 
to  which  barnyard  manure  and  bone  meal  had  been  added.  Especial 
care  was  taken  to  keep  all  plants  under  the  same  conditions  of  tempera- 
ture, moisture,  and  light  both  before  and  after  inoculation. 

On  the  wheat  plants  which  were  grown  on  very  heavily  fertilized 
soil  the  infection  was  clearly  more  severe.  The  infected  areas  were 
very 'large  as  were  also  the  individual  pustules.  The  most  severely 
attacked  plants  were  in  one  pot  which  had  been  fertilized  with  manure 
and  bone  meal.  Aside  from  this  one  pot,  however,  there  was  but  little 
variation  among  the  fertilized  pots.  The  infection  on  the  check  plants 
remained  inferior,  although  it  was  very  vigorous. 

Similar  results  were  obtained  from  einkorn  and  lumillo  plants.  In 
the  case  of  these  two  forms  the  plants  in  the  manure-and-bone  series 
developed  the  worst  rust  attack,  the  manure  was  next,  and  the  plants 
in  ordinary  soil  were  more  lightly  attacked.  It  should  be  remarked  that 
the  differences-  were  not  strikingly  sharp,  although  they  were  quite 
apparent.  Emmer  and  Arnautka  gave  no  distinct  results.  There  was 
a  great  deal  of  variation  in  the  individual  pots  and  no  one  series  stood 
out  clearly  from  the  other  two. 

The  Kubanka  and  Khapli  plants  showed  some  differences.  The 
character  of  infection  is  very  different  from  that  of  the  other  forms. 


RUST-RESISTANT  VARIETIES  OF  WHEAT 


37 


In  the  case  of  the  former  the  plants  grown  in  pots  to  which  barnyard 
mauure  had  been  added  were  most  severely  attacked,  while  there  was 
little  difference  between  those  grown  in  loam  and  those  manured  with 
both  barnyard  manure  and  bone  meal.  Khapli  plants  grown  in  soil 
fertilized  with  both  manure  and  bone  were  fairly  successfully  infected. 
Areas  of  the  leaf,  one  centimeter  in  length,  were  sometimes  infected, 
and  pustules  as  big  as  a  pin  head  were  developed.  There  was  little  dif- 
ference between  the  other  pots,  the  infection  being  somewhat  milder 
than  in  the  heavily  fertilized  ones 

It  is  quite  probable  that  there  was  available  in  the  rich  loam  very 
nearly  all  of  the  plant  food  the  plants  were  capable  of  utilizing.  This 
would  account  for  the  fact  that  the  differences  were  not  always  greater. 
On  the  whole  it  might  be  concluded  that  very  heavy  fertilization  is 
somewhat  conducive  to  increased  severity  of  attack  on  very  resistant 
varieties  as  well  as  on  susceptible  forms. 

Since  the  check  plants  in  the  trials  just  described  were  grown  un- 
der such  favorable  conditions,  it  was  determined  to  grow  the  checks  in 
poorer  soil  in  the  next  series.  Therefore  they  were  planted  in  mod- 
erately fine  sand  (S)  (see  table  below)  to  which  but  a  very  slight 
amount  of  leaf-mold  had  been  added.  Nitrogen  (N)  was  added  to 
another  series  in  the  form  of  calcium  nitrate,  to  a  third  was  added  cal- 
cium phosphate  (P),  and  the  fourth  received  both  calcium  phosphate 
and  calcium  nitrate  (P  and  N).  The  salts  were  applied  in  water.  The 
plants  were  watered  three  times  with  distilled  water  containing  the 
proper  salt  or  salts,  at  the  rate  of  3  grams  per  500  cc.  The  same  varie- 
ties were  used  as  were  used  in  the  preceding  series. 

Observations  on  results  gave  the  following  order  of  virulence,  the 
first  being  most  virulent  and  the  others  arranged  on  the  same  basis. 
Two  observers  took  notes  with  the  following  results : 

EFFECT  OF  FERTILIZERS  ON  VIRULENCE  OF  RUST  ATTACK 


Grain 

Order  of  Virulence 

1 

2 

3 

4 

Einkorn 

N 

P  and  N            P 

S 

Emmer 

PandN 

N 

p 

S 

Kubanka 

PandN 

N 

P 

S 

Khapli 

PandN 

N 

P 

S 

Arnautka 

N 

S 

P 

PandN 

lumillo 

N 

P 

S 

PandN 

The  somewhat  conflicting  results  suggested  the  desirability  of  an- 
other trial.  Four  series  were  arranged  as  follows:  Pure  sand  (S), 
ordinary  field  soil  (O),  sand  plus  nitrogen  (N),  and  sand  plus  phos- 
phorus (P).  The  nitrogen  and  phosphorus  were  added  as  in  the  pre- 
ceding experiment.  Three  persons  working  independently  made  ob- 


38 


A  STUDY  IN  CEREAL  RUSTS 


servations,  but  there  was  no  great  difference  of  opinion  in  any  case. 
The  order  in  which  different  observers  placed  them  was  sometimes  dif- 
ferent, showing  that  there  was  sometimes  little  choice  among  the 
various  pots.  The  results  are  given  in  the  following  table : 

EFFECT  OF  FERTILIZERS  ON  VIRULENCE  OF  RUST  ATTACK 


Order  of  Virulence 

Grain 

Remarks 

1 

2 

3 

4 

Emmer 

N 

P 

S 

0 

Kubanka 

S 

P 

N 

0 

lumillo 

N 

0 

P 

S 

Khapli 

S 

N 

P 

O 

Arnautka 

P 

S 

N 

0 

Wheat 

S 

N 

P 

O 

Einkorn 

N 

P 

S 

O 

Einkorn 

P 

N 

S 

0 

Jttle  difference  between  N  and  P 

Emmer 

N 

S 

P 

0 

Little  difference  between  P  and  S 

Kubanka 

S 

P 

N 

0 

Distinct 

lumillo 

N 

P 

O 

S 

Little  difference  between  P  and  N 

Khapli 

S 

N 

P 

0 

Little  difference  between  P  and  N 

Arnautka 

P 

N 

S 

0 

Little  difference  between  N  and  S 

Wheat 

N 

S 

P 

0 

Little  difference  between  S  and  P 

In  this  experiment  the  two  most  resistant  forms  available  for  study 
were  watched  very  carefully.  Both  Kubanka  2094  and  Khapli  proved 
to  be  very  resistant  even  when  very  highly  fertilized.  It  is  a  rather 
striking  fact  that  in  both  cases  plants  grown  in  sand  showed  a  slightly 
more  virulent  infection.  The  differences  were  not  great.  In  fact,  it 
was  often  hard  to  decide  which  plants  were  most  severely  affected.  It 
was  observed  that  plants  which  had  been  under  the  most  favorable  con- 
ditions for  infection  were  most  severely  attacked  regardless  of  the  fer- 
tilizer used.  The  difference  in  conditions  was  due  to  the  fact  that  there 
were  not  enough  bell  jars  to  cover  all  the  plants,  so  some  were  placed 
in  tubs  containing  water  on  the  bottom.  They  were  then  covered  with 
pieces  of  glass.  The  films  of  moisture  were  not  so  persistent  here  as 
under  the  jars,  and  the  difference  in  the  amount  of  rust  was  quite 
marked.  This  was  taken  into  account  in  determining  results  and  mak- 
ing comparisons. 

If  these  plants  had  been  allowed  to  grow  longer  it  is  quite  prob- 
able that  those  fertilized  with  nitrogen  would  have  become  more  se- 
verely rusted  while  those  fertilized  with  phosphorus  would  have  been 
slightly  less  severely  affected.  This  would  seem  to  be  due  not  to  the 
specific  action  of  the  chemicals  on  the  rust  fungus  but  rather  to  their 
effect  on  the  general  condition  of  the  plant,  and,  in  the  field,  on  the 


RUST-RESISTANT  VARIETIES  OF  WHEAT  39 

immediate  atmospheric  conditions.  The  direct  effect  of  chemicals  in 
the  soil  on  the  amount  of  rust  on  resistant  varieties  is  not  great,  only  a 
slight  quantitative  difference  being  apparent. 

In  order  to  determine  by  more  accurate  methods  whether  or  not 
there  was  a  direct  effect  of  substances  in  the  soil  on  the  amount  of 
rust  (1)  fungicides  were  put  into  nutrient  media,  and  (2)  certain 
nutrient  salts  were  used  in  amounts  varying  from  deficiency  to  excess. 

Sach's  modified  culture  solution  was  used  and  to  this  one  per  cent 
of  agar  was  added.  The  series  was  arranged  as  follows,  the  amounts 
of  nitrate  and  phosphate  being  the  variables : 

I.     Potassium  nitrate;  2  grams  per  1,000  cc. 
II.     Calcium  phosphate;  3  grams  per  1,000  cc. 

III.  Potassium  nitrate ;  .05  grams  per  1,000  cc. 

IV.  Calcium  phosphate;  .075  grams  per  1,000  cc. 
Minnesota  No.  163  wheat  was  used.    After  inoculation  plants  were 

all  placed  under  bell  jars  and  kept  under  uniform  conditions.  Arranged 
according  to  virulence,  the  most  severely  affected  being  placed  first, 
they  would  be  arranged  as  follows:  III,  II,  IV,  I. 

This  was  tried  a  second  time  with  exactly  the  same  results.  The 
plants  appeared  about  the  same,  all  growing  fairly  well  in  the  agar. 
All  were  well  infected,  producing  a  fairly  large  number  of  large, 
healthy  pustules.  They  were  kept  three  weeks  after  inoculation  and 
by  this  time  there  was  not  much  difference  between  II,  IV,  and  I,  but 
III  was  still  much  more  virulently  attacked.  It  seems,  therefore,  that 
an  excess  of  nitrogen  does  not  necessarily,  in  itself,  cause  an  increase  in 
the  amount  of  rust  and  an  excess  of  phosphorus  does  not  affect  it  very 
appreciably. 

The  effect  of  excluding  nitrogen  and  phosphorus  was  next  tried. 
Sach's  modified  medium  plus  one  per  cent  of  agar  was  again  used  and 
in  I  no  potassium  nitrate  was  added  while  calcium  phosphate  was  ex- 
cluded from  II.  The  plants  in  I  were  lighter  colored  from  the  first 
than  either  those  in  II  or  the  checks.  They  were  inoculated  six  days 
after  planting.  A  good,  vigorous  infection  resulted,  the  plants  in  I 
being  slightly  more  severely  attacked  than  those  in  II.  The  leaves  of 
I  began  to  turn  yellow  after  three  weeks,  and  the  rust  did  not  spread 
farther.  The  check  plants  were  more  severely  attacked  than  those  in 
either  I  or  II.  Here  again,  however,  the  differences  were  not  very 
great.  There  was  a  slight  quantitative  difference  but  qualitatively  there 
was  scarcely  any  difference.  This  is  in  keeping  with  Ward's  conclu- 
sions reached  after  his  work  on  mineral  starvation,  reference  to  which 
has  already  been  made. 

An  attempt  was  then  made  to  determine  whether  it  was  possible 
to  confer  immunity  by  means  of  various  salts.  Copper  sulfate,  copper 
carbonate  and  iron  sulfate  were  used  in  strengths  varying  from  1  to 


40  A  STUDY  IN  CEREAL  RUSTS 

10,000  to  1  to  2,000.  They  were  added  to  Sach's  medium  in  these  pro- 
portions. Minnesota  No.  163  was  used  for  all  the  trials.  Copper  sul- 
fate  could  not  be  added  in  larger  amounts  than  1  to  5,000,  since  it 
stunted  the  plants  when  more  was  added.  Copper  carbonate  could  not 
well  be  used  in  greater  concentration  than  1  to  2,000.  Iron  sulfate 
did  not  dwarf  the  plants  when  used  at  the  rate  of  1  to  2,000.  None  of 
the  solutions  used  diminished  the  amount  of  rust  very  appreciably  when 
used  in  such  concentration  as  to  permit  of  normal  development  of  the 
host  plant.  There  was  a  slightly  smaller  amount  of  rust  on  plants 
grown  in  the  medium  to  which  copper  sulfate  had  been  added  in 
amounts  of  1  to  4,000  and  1  to  2,000.  However,  a  very  good  infection 
was  secured  on  all  of  them,  even  on  those  which  never  grew  to  a  greater 
height  than  one  inch.  Neither  was  there  any  less  mildew  on  any  of 
the  plants.  None  of  these  substances,  apparently,  can  immunize  wheat 
against  rust,  even  when  used  in  such  concentration  as  to  dwarf  the 
plants  to  one-sixth  their  normal  size. 

These  experiments  show  that  in  the  case  of  Puccinia  gramims, 
since  it  is  a  very  highly  specialized,  obligate  parasite,  there  is  a  very 
intimate  relationship  between  host  and  parasite,  and  whatever  is  con- 
ducive to  the  health  of  host  is  ordinarily  conducive  to  the  vigorous  de- 
velopment of  the  parasite  also.  This  applies  not  only  to  susceptible 
forms  but  also  to  forms  uncongenial  to  a  biologic  form  and  to  resistant 
varieties. 

THE  NATURE  OF  RESISTANCE 

The  work  of  Cobb,  Eriksson,  Ward,  and  others  on  the  nature  of 
resistance  has  already  been  mentioned.  The  theory  which  Ward  de- 
duced from  his  extensive  work  on  parasitism  was  that  there  are  en- 
zymes or  toxins  and  antitoxins  produced  by  host  or  parasite  or  both. 
His  work  on  "A  Lily  Disease"  (1888)  showed  that  in  all  probability 
Botrytis  secretes  an  enzyme  which  enables  it  to  live  more  successfully 
on  the  host.  Pfeffer  had  already  given  the  name  chemotaxis  to  the  at- 
traction certain  substances  seemed  to  have  for  certain  growing  plant 
parts.  Miyoshi  (1894,  p.  21)  claimed  to  have  been  able  to  observe  a 
very  definite  chemotropism  when  a  Tradescantia  leaf  was  injected 
with  a  wheat-leaf  decoction  and  then  inoculated  with  Uredo  linearis 
(Puccinia  graminis).  The  same  author  decided  (1895)  that  a  large 
number  of  fungi  responded  to  chemical  attraction.  Massee  (1904, 
p.  7)  attached  a  great  deal  of  significance  to  chemotaxis.  He  asserted 
that  infection  depended  on  the  presence  in  the  plant  cell  of  positive 
chemotactic  substances  and  further  that  "in  the  future  we  shall  be  justi- 
fied in  defining  an  immune  plant  as  an  individual  in  which  the  positive 
chemotactic  substance,  necessary  for  facilitating  the  entrance  of  the 
germ-tubes  of  a  given  parasitic  fungus  into  its  tissues,  is  absent."  On 


RUST-RESISTANT  VARIETIES  OF  WHEAT  41 

the  other  hand  Errera  (1892,  p.  373)  contends  that  so-called  chemotro- 
pism  is  in  many  cases  merely  positive  or  negative  hydrotropism.  Ful- 
ton (1906,  pp.  81-107)  says  that  there  is  no  definite  chemotropic  re- 
sponse on  the  part  of  fungi.  Nutrient  solutions  cause  marked  growth, 
in  his  opinion,  but  cause  no  definite  turning  of  hyphae  in  their  direc- 
tion. Hydrotropism,  however,  was  observable. 

The  behavior  of  the  germ  tube  of  Puccinia  graminis  and  its  en- 
trance into  the  host  plant  has  been  described  and  figured  by  various 
authors.  Ward  (1881-1,  p.  217)  figures  it  as  forming  a  slight  swell- 
ing and  then  growing  directly  into  the  tissues 'of  the  host.  Bolley 
( 1889,  p.  14)  shows  the  germ  tube  growing  directly  through  the  stom- 
atal  opening  and  branching  out  between  the  mesophyll  cells.  This  has 
never  been  seen  by  the  writer.  An  appressorium  always  formed  in  all 
cases  of  infection  observed.  Pole  Evans  (1907,  p.  445)  describes  and 
figures  normal  infection  quite  completely. 

A  considerable  amount  of  work  has  also  been  done  on  determining 
the  fate  of  germ  tubes  when  they  are  produced  on  immune  host  plants. 
Klebahn  noted  (1896,  p.  263)  that  sporidia  of  Puccinia  convallariae- 
digraphidis  could  bore  through  the  epidermal  walls  of  Polygonatum 
multifiorum,  an  uncongenial  host,  but  that  the  germ  tubes  developed  no 
further.  He  concludes  that  infection  is  of  the  nature  of  a  conflict  be- 
tween host  and  parasite.  Ward  (1901  and  1902)  showed  in  connection 
with  Puccinia  dispersa  on  bromes  that  the  germ  tube  might  enter  and 
cause  normal  infection,  the  mycelium  might  develop  and  never  produce 
pustules,  or  the  tissues  of  the  host  plant  might  be  killed  very  early, 
thus  precluding  the  possibility  of  much  development  on  the  part  of 
the  fungus.  The  same  author  further  shows  (1904,  p.  29)  that  in 
normal  infection  of  a  susceptible  species  of  Bromus  the  host  cells 
retain  life  for  a  surprisingly  long  time.  Miss  Gibson  (1904)  examined 
the  leaves  of  a  large  number  of  plants,  widely  separated  taxonomically, 
which  had  been  inoculated  with  spores  of  I] redo  chrysanthemi.  She 
found  that  the  germ  tubes  might  enter  the  plant  tissues  very  readily 
but  never  formed  any  haustoria  and  consequently  no  pustules. 
Furthermore  the  hyphae  usually  died  when  t^ey  came  in  contact  with 
a  cell.  On  resistant  varieties  of  Chrysanthemum  it  was  found  that 
haustoria  might  develop,  but  areas  of  host  tissue  in  the  neighborhood 
of  the  hyphae  were  killed,  thus  preventing  the  further  spread  of  the 
fungous  mycelium.  Her  conclusion  is  to  the  effect  that  when  the 
germ  tube  of  a  uredine  fungus  enters  any  but  its  proper  host  plant  a 
struggle  goes  on,  resulting  in  the  death  of  the  host  locally  and  of  the 
parasite.  The  closer  the  relationship  between  the  plant  and  the  proper 
host  of  the  rust  the  longer  and  more  extensive  will  be  the  struggle. 
Salmon  (1905)  found  that  when  barley  was  inoculated  with  spores 
of  Erysiphe  graminis  from  wheat  incipient  haustoria  might  be  formed 


42  A  STUDY  IN  CEREAL  RUSTS 

in  the  cells,  but  that  they  became  disorganized  within  a  very  few  days. 
He  attributed  this  to  defective  symbiotic  relations  between  host  and 
parasite.  Miss  Marryat  (1907)  showed  that  Puccinia  glumarum  when 
grown  on  a  semi-immune  host  plant  killed  local  areas  of  the  host,  sent 
out  but  few  haustoria,  and  never  developed  any  but  small  or  abortive 
pustules. 

It  is  a  matter  of  common  observation  that  in  dealing  either  with 
cereals  uncongenial  to  a  given  biologic  form  of  Puccinia  graminis  or 
with  varieties  of  wheat  resistant  to  Puccinia  graminis  tritici  flecks  are 
often  visible  after  inoculation,  but  no  pustules,  or  only  small  ones,  are 
produced.  Examples  of  this  are  shown  in  Plate  I,  A  and  Plate  VI,  A. 
All  degrees  of  this  killing  can  be  observed.  The  more  readily  the  rust 
infects  a  plant  the  less  likely  are  these  dead  areas  to  appear.  When 
Puccinia  graminis  tritici  is  put  on  Minnesota  No.  163  wheat,  pustules 
are  formed  in  great  abundance,  but  the  leaf  tissues  remain  alive  for  a 
long  time.  When,  on  the  other  hand,  resistant  forms  such  as  Kubanka 
2094  or  Khapli  are  inoculated,  areas  of  the  leaf  are  killed  outright ;  and 
if  pustules  are  formed  at  all  they  are  very  small.  In  extreme  cases  of 
incompatibility  such  as  is  found  between  Puccinia  graminis  avenae 
and  wheat,  the  leaf  area  involved  is  usually  so  small  that  no  indications 
of  it  can  be  seen  with  the  unaided  eye. 

HlSTOLOGICAL  DETAILS  OF  INFECTION 

In  order  to  determine  the  behavior  of  germ  tubes  in  susceptible 
and  nearly  immune  forms,  leaves  of  Minnesota  No.  163  and  of  Khapli 
were  inoculated  with  Puccinia  graminis  tritici.  Minnesota  No.  163  was 
also  inoculated  with  Puccinia  graminis  avenae.  They  were  then  placed 
in  a  pan  of  water  under  bell  jars  for  48  hours.  Leaves  were  selected 
and  killed  every  24  hours,  beginning  with  the  first  day. 

For  killing,  aceto-alcohol,  medium  chromo-acetic  acid,  and  Flem- 
ming's  weaker  killing-fluid  were  used.  The  leaves  were  embedded 
in  the  usual  manner  and  sectioned  from  five  to  ten  microns  thick. 
For  staining,  Haidenhain's  iron-alum  haematoxylin  and  orange  G, 
Erlich's  haematoxylin,  Gram's  stain  and  eosin,  Delafield's  haematoxylin 
used  according  to  Durand's  method,  fuchsin  and  methyl  green  and  the 
safranin,  gentian  violet,  orange  G.  combination,  used  according  to 
Harper's  modification  of  Flemming's  method,  were  used.  The  last 
named  gave  the  best  results. 

INFECTION  OF  MINNESOTA  NO.   163 

At  the  end  of  24  hours  many  of  the  spores  have  usually  germ- 
inated, sending  out  long  germ  tubes,  although  some  apparently 
germinate  later.  Two  tubes  may  be  sent  out  from  the  same  spore, 
but  usually  one  develops  more  vigorously  than  the  other.  The  tube 
usually  follows  the  epiderm  closely;  swellings  are  often  found  above 


RUST-RESISTANT  VARIETIES  OF  WHEAT  43 

the  wall  between  two  epidermal  cells  (Plate  VII,  3).  In  these  swell- 
ings, which  appear  very  much  like  very  young  appressoria,  the  pro- 
toplasm often  aggregates  more  densely  than  in  the  other  parts  of  the 
tube.  The  germ  tube  may  often  follow  the  epiderm  for  considerable 
distances,  sometimes  for  the  length  of  15  epidermal  'cells,  before  a 
definite  appressorium  is  formed.  Nearly  always  when  a  stoma  is 
reached  the  tube  forms  a  very  definite  swelling  (Plate  VII,  1)  which 
constitutes  an  appressorium.  The  protoplasmic  contents  of  practically 
the  entire  germ  tube  are  concentrated  in  this  appressorium.  It  dips 
down  into  the  stomatal  opening  and  a  fine  protoplasmic  process  is  sent 
through  to  the  substomatal  space.  Here  a  definite  swelling  takes 
place,  forming  the  substomatal  vesicle  (Plate  VII,  4  and  5).  Appar- 
ently, in  some  cases,  the  vesicle  develops  no  further.  In  the  great 
majority  of  cases,  however,  the  protoplasm  aggregates  in  it  and 
infection-threads  are  sent  out.  Very  often  these  infection-threads 
follow  closely  along  under  the  epiderm  cells  and  send  small  knoblike 
or  sometimes  flattened  haustoria  into  the  host  cells.  Branching  then 
takes  place  among  the  cells  of  the  leaf,  many  haustoria  being  sent  out 
and  the  hyphae  developing  very  rapidly.  The  threads  may  grow 
directly  across  the  substomatal  space  and  branch  between  the  mesophyll 
cells  (Plate  VII,  6).  This  does  not  seem  to  be  the  usual  method, 
however.  Nuclear  division  takes  place  rapidly  to  keep  pace  with  the 
growth  of  the  hyphae.  Frequently  a  number  of  nuclei  are  found  in  a 
single  cell.  The  entire  hypha  often  retains  its  protoplasm  for  a  con- 
siderable length  of  time.  Ordinarily  the'  tip  only  remains  densely 
protoplasmic  while  the  rest  of  the  hypha  becomes  much  vacuolated. 
Very  long  hyphae  are  often  found  growing  very  vigorously  but  appar- 
ently not  sending  out  haustoria.  They  seem  to  be  in  the  nature  of 
distributive  filaments.  The  fungus  does  not  seem  to  spread  very  far 
from  the  point  of  infection.  When  large  areas  of  the  leaf  are  involved, 
a  number  of  points  of  entry  can  nearly  always  be  found.  On  the 
fourth  day  an  infected  area  is  usually  well  filled  with  much  branched 
hyphae.  About  this  time  wefts  begin  to  be  formed,  the  protoplasm 
aggregating  in  the  tips  of  the  hyphae.  A  dense  mass  of  filaments  is 
formed  just  beneath  the  epiderm  and  the  epidermal  cells  are  wedged 
apart.  The  large,  upright  fungus  cells,  which  show  the  binucleate 
condition  very  clearly,  begin  to  form  uredospores.  This  is  well  under 
way  on  the  fifth  day  and  by  the  sixth  or  seventh  day  the  epiderm  has 
been  completely  ruptured  while  spores  are  being  shed  in  great  pro- 
fusion. It  is  interesting  to  note  that  the  host  cells,  which  are  often 
half  filled  with  large  knoblike,  filamentous  or  coiled  haustoria,  are 
usually  still  quite  healthy  at  the  time  pustule  formation  begins. 

The  whole  appearance  of  both  fungus  and  host  during  the  first 
few  days  after  infection  indicates  a  fairly  perfect  relation  between 


44  A  STUDY  IN  CEREAL  RUSTS 

the  two.  The  fungus  flourishes  vigorously  while  for  a  considerable 
length  of  time  the  host  cells,  even  in  the  infected  area,  are  apparently 
quite  healthy.  In  no  case  does  there  seem  to  be  an  extensive  killing  of 
host  tissue. 

INFECTION   OF   KHAPLI 

Spore  germination  of  course  occurs  normally.  The  germ  tubes 
grow  along  the  surface  of  the  host  epiderm  cells  in  the  same  manner 
as  do  those  on  Minnesota  No.  163.  The  formation  of  appressoria 
takes  place  in  an  entirely  normal  manner.  The  stimulus  causing  entry 
into  the  stomatal  slit  is  present,  the  method  of  entrance  being  sub- 
stantially the  same  as  in  Minnesota  No.  163.  Apparently  the  vesicle 
sometimes  fails  to  send  out  infection  threads  but  merely  remains 
directly  beneath  the  stomatal  slit  and  becomes  vacuolated.  It  may  send 
out  numerous,  short,  club-shaped  branches  all  of  which  soon  become 
vacuolated  and  never  send  out  any  haustoria  into  the  host  cells.  From 
the  beginning  of  growth  in  the  host  it  is  easily  discernible  that  the 
vigor  of  the  hyphae  is  not  nearly  so  great  as  is  the  case  with  those 
growing  in  Minnesota  No.  163.  There  is  a  greater  tendency  for  the 
tips  of  infection  threads  to  round  up,  become  vacuolated,  and  never 
develop  further. 

Fairly  successful  infection,  however,  may  take  place.  Infection 
threads  may  be  sent  out  just  under  the  epiderm  or  directly  across  the 
substomatal  space  (see  Plate  VIII,  3  and  5).  Haustoria,  attached  to 
the  hypha  by  delicate  sterigmata,  are  sent  into  the  cells  and  the  hypha 
grows  fairly  well.  Sometimes  many  incipient  infection  threads  are 
formed  from  a  single  vesicle,  only  one  developing  (Plate  VIII,  5). 
Shortly  after  infection  threads  are  sent  out  the  vesicle  usually  dies. 

The  infection  threads  are  not  always  successful  in  sending 
haustoria  into  the  host  cells.  When  the  hypha  comes  into  contact 
with  the  cells  the  protoplast  of  the  latter  often  shrinks  back  from  the 
wall,  the  nucleus  shows  definite  signs  of  disintegration,  the  chloroplasts 
are  apparently  lost,  and  the  entire  cell  dies.  The  hypha  may  die  also, 
or  it  may  grow  and  kill  other  cells.  However,  it  usually  eventually 
succumbs.  Typical  examples  of  this  will  be  seen  on  Plate  VIII,  6  and 
7.  It  will  be  noticed  that  at  6  the  cell  on  which  the  hypha  is  abutting 
is  apparently  dead,  the  chloroplasts  have  disappeared,  and  the  nucleus 
is  disintegrating.  At  7  this  has  taken  place  in  only  a  part  of  the  cell. 
It  sometimes  happens  that  one  branch  of  a  hypha  is  fairly  successful 
while  another  may  never  develop  to  any  extent  at  all. 

Whether  or  not  the  host  cells  are  killed  within  a  short  time  after 
the  hyphae  come  in  contact  with  them,  infection  does  not  appear  to 
be  normal.  The  hyphae  may  grow  fairly  well,  but  never  as  vigorously 
as  in  Minnesota  No.  163.  Haustoria  may  be  sent  out  in  fair  abundance 


RUST-RESISTANT  VARIETIES  OF  WHEAT  45 

(Plate  IX,  1,  2,  and  4).  The  hyphal  tips  may  branch  very  profusely 
but  they  become  vaculoated  very  early  in  most  cases  and  appear  un- 
thrifty. It  requires  8  or  9  days  for  the  stage  of  infection  to  be 
reached  on  Khapli  that  is  reached  in  3  or  4  days  in  susceptible  varieties. 
Short,  thick,  or  rounded  hyphal  segments  are  quite  common,  those 
at  the  end  of  a  branch  often  containing  as  many  as  5  or  6  nuclei,  some 
of  which  appear  to  be  disintegrating. 

The  tips  of  hyphae  naturally  die  when  a  group  of  host  cells  among 
which  they  are  growing  is  killed.  However,  they  may  disintegrate 
without  having  first  killed  host  cells  (Plate  IX,  1,  2,  3,  and  4).  There 
may  be  many  variations.  The  hyphae  may  not  send  out  haustoria  and 
die  in  consequence,  or,  even  if  they  do  send  them  into  the  cells,  death 
may  occur.  Branches  of  hyphae  which  have  sent  haustpria  into  host 
cells  frequently  become  vacuolated  and  gradually  die,  or  the  pro- 
toplasmic contents  may  change  to  granular,  deep-staining  masses. 
The  whole  appearance  suggests  fungous  hyphae  growing  in  an  un- 
favorable nutrient  solution. 

In  about  11  or  12  days  a  distinct  tendency  toward  the  formation 
of  hyphal  wefts  can  be  observed.  These  vary  greatly  both  in  size 
and  position.  They  may  be  mere  aggregations  directly  under  the 
epiderm  or  deeper  down  in  the  tissues;  or  they  may  begin  to  wedge 
the  epiderm  cells  apart  after  the  manner  of  young  pustules.  Often 
small,  unruptured  pustules  are  formed  in  which  there  is  a  fairly  large 
number  of  abortive  spores.  The  pustules  may  rupture  the  epiderm 
but  they  are  always  extremely  small,. and  as  a  rule  the  spores  are 
small.  The  average  size  of  a  large  number  of  spores  measured  was 
29.69  x  20.68  /*,  whereas  the  average  size  of  the  wheat  rust  spores 
which  were  used  in  making  the  inoculations  was  35.38x21.39  yw. 

After  about  20  days  practically  all  the  host  cells  and  a  large 
number  of  the  hyphae  in  an  infected  region  are  dead.  Haustoria  may 
be  present  in  the  host  cells  in  fairly  large  numbers,  but  most  of  them 
are  dead,  their  protoplasmic  contents  having  broken  up  into  granular, 
deep-staining  masses.  The  nuclei  of  the  host  cells  are  often  dis- 
integrating also.  The  infection  by  this  time,  and  usually  earlier,  has 
completely  run  its  course.  Comparatively  very  few  spores  have  been 
produced,  and,  under  natural  conditions,  secondary  infections  would 
probably  not  occur  to  any  extent. 

THE    COURSE    OF    INFECTION    IN    OTHER    RESISTANT    FORMS 

Substantially  the  same  sequence  of  events  occurs  in  other  resistant 
forms,  such  as  Arnautka  288,  Kubanka  2094,  emmer  1522,  einkorn  2433 
(sometimes),  and  in  such  cases  as  the  infection  of  rye  by  barley  rust. 
The  differences  seem  to  be  in  degree  rather  than  in  kind.  In  the  cases 
of  emmer  and  einkorn  the  killing  of  host  cells  is  rarely  found  but 


46  A  STUDY  IN  CEREAL  RUSTS 

unsuccessful  attempts  to  form  pustules  are  often  noticed.  The 
sequence  of  events  in  Arnautka  288  and  Kubanka  2094  is  quite  similar 
to  that  in  Khapli.  These  are  semi-immune  forms  in  which  the  contest 
between  host  and  parasite  is  somewhat  prolonged.  Fatalities  occur  on 
both  sides  but  not  in  sufficient  number  to  render  infection  absolutely 
unsuccessful.  It  is,  therefore,  essential  to  note  the  sequence  in  such 
extreme  cases  of  almost  total  immunity  as  are  furnished  by  wheat 
when  inoculated  with  Puccinia  graminis  avenae. 

MINNESOTA    NO.     163    INOCULATED    WITH    PUCCINIA    GRAMINIS    AVENAE 

It  will  be  recalled  that  very  rarely  indeed  does  successful  infection 
follow  inoculation  of  wheat  with  Puccinia  graminis  avenae.  Long 
germ  tubes  are  sent  out  by  spores  germinating  on  the  surface  of  the 
leaf.  These  follow  the  epiderm,  dipping  into  depressions  in  an  entirely 
normal  manner.  Appressoria  are  found,  a  small  neck  grows  through 
the  stomatal  slit  and  the  substomatal  vesicle  is  formed.  This  vesicle 
sometimes  sends  out  only  very  small  knoblike  branches  which  soon 
die,  or  branches  may  be  sent  out  and  very  few  hausteria  produced. 

The  vesicle  often  sends  out  many  short,  knoblike  branches, 
appearing  almost  like  elongated  amoebae.  These  do  not  appear 
vigorous  after  a  few  days.  The  epiderm  cells  in  the  immediate  vicinity 
appeared  to  be  killed.  If  definite  infection  threads  are  sent  out  they 
never  grow  very  long  but  kill  two  or  three  host  cells  and  then  stop 
growing. 

The  difference  between  conditions  in  such  a  case  as  this  and 
such  a  one  as  Khapli  is  apparently  in  degree  only.  Whereas  in  Khapli 
the  fungus  might  develop  to  a  certain  extent,  thus  involving  fairly 
large  leaf  areas,  in  such  an  extreme  case  of  immunity  as  is  presented 
by  wheat  to  oat  rust  only  a  few  host  cells  are  involved  and  the  contest 
between  host  and  parasite  is  short  and  decisive,  only  a  very  few  host 
cells  being  killed;  and  the  hyphae  seldom  develop  sufficiently  to  give 
any  external  evidence  that  the  germ  tube  has  even  entered. 

As  to  the  fundamental  causes  for  these  facts,  only  speculation  is 
possible.  It  would  seem  reasonable  to  suppose  either  that  there  was 
a  lack  of  an  attracting  substance  or  the  presence  of  a  deleterious  sub- 
stance. Massee's  chemotaxis  theory  has  already  been  mentioned. 
Chemotaxis  or  its  absence  would  hardly  explain  the  phenomena  men- 
tioned, since  the  fungus  succeeds  in  effecting  an  entrance  into  even  the 
most  immune  forms.  The  evidence  would  rather  seem  to  favor  the 
view  that  the  whole  problem  is  one  of  toxins  in  host  or  parasite  or, 
very  probably,  in  both.  In  some  cases  the  host  is  apparently  hyper- 
susceptible,  while  further  study  may  prove  that  there  is  in  other  cases 
a  real  resistance.  Careful  biochemical  investigation  alone  can  settle 
this  question  definitely. 


RUST-RESISTANT  VARIETIES  OF  WHEAT  47 

Certainly,  however,  there  can  be  no  question  of  a  certain  antagon- 
ism between  host  and  parasite  when  one  observes  phenomena  such  as 
are  illustrated  in  Plate  VIII,  6.  This  is  in  remarkably  sharp  contrast 
with  the  apparent  congeniality  exhibited  in  such  cases  as  are  shown 
at  6,  7,  and  8  in  Plate  VII.  Antagonism  would  seem  to  be  explicable 
at  present  only  by  the  toxin  or  enzyme  theory.  The  recent  work  of 
Bolley  (1908  and  1909),  Pole  Evans  (1909  and  1911),  McAlpine 
(1910),  Freeman  and  Johnson  (1911),  and  BifTen  (1912)  indicates 
clearly  that  immunity  and  resistance  are  concepts  which,  from  the 
very  nature  of  their  variability  and  sometimes  apparent  capriciousness, 
must  be  cautiously  discussed.  At  least  one  substance,  commonly 
found  in  plants,  has  been  found  by  Cook  and  Taubenhaus  (1911,  pp. 
40  and  43)  to  be  toxic  to  certain  rusts  of  the  genus  Uromyces. 

Whatever  the  immediate  instruments  governing  congeniality  or 
antagonism,  the  fundamental  facts  brought  out  quite  clearly  in  the 
results  described  in  the  present  investigation  have  a  bearing  on  the 
practical  and  theoretical  questions  involved  in  the  problem  of  pre- 
venting cereal  rusts  and  the  breeding  of  rust-resisting  varieties.  Ex- 
ternal morphology  as  pointed  out  by  Ward  (1902-1)  for  brome  rusts, 
Salmon  (1905-2)  for  mildews,  and  Biffen  (1907)  for  yellow  rust  is 
also  of  very  slight  importance  in  the  immunity  of  cereal  varieties  to 
stem  rust.  No  observed  facts  in  intimate  histology,  moreover,  give 
any  clue  to  resistance.  In  the  absence  of  biochemical  information 
concerning  the  activities  of  invading  hyphae  and  invaded  host  tissues, 
actual  performance  alone  can  be  depended  upon  as  a  safe  criterion 
in  the  development  of  resistant  forms  or  immune  varieties.  This  is 
all  the  more  true  since  Pole  Evans  (1911)  has  found  that  a  hybrid 
wheat  produced  by  crossing  rust-immune  and  rust-susceptible  wheats 
may  rust  quite  badly  and  be  capable  of  causing  infection  of  the  immune 
parent  and  a  more  severe  attack  of  rust  on  the  susceptible  parent 
variety  than  rust  from  that  variety  itself  will  cause.  The  production 
of  flecks  and  dead  areas  on  an  inoculated  plant  is  a  character  of 
possible  use  in  indicating  at  least  a  semi-immunity. 

There  does  not  seem  to  be  any  obvious  constant  correlation  be- 
tween immunity  and  other  observable  characters,  as  for  instance 
drought-resistance.  Although  the  immune  varieties  of  wheat  used 
in  this  investigation  are  drought-resistant,  it  is  also  a  well-estab- 
lished fact  that  other  drought-resistant  wheats  such  as  Kubanka  1516 
are  very  susceptible.  Moreover,  the  antagonism  exhibited  by  Minne- 
sota No.  163  wheat  toward  Puccinia  graminis  avenae  and  by  rye 
toward  Puccinia  graminis  hordei  does  not  differ  fundamentally  from 
that  exhibited  by  Khapli  toward  Puccinia  graminis  tritici'and  in  the 
first  two  cases  a  correlation  with  drought-resistance  is  out  of  the 
question. 


48  A  STUDY  IN  CEREAL  RUSTS 

That  the  important  scientific  questions  involved  in  the  specializa- 
tion of  biologic  forms  and  that  of  rust-resistant  varieties  of  wheat 
are  essentially  the  same  seems  obvious.  The  same  phenomena  can  be 
observed  in  both ;  there  are  various  degrees  of  resistance  and  suscep- 
tibility in  both  and  a  thorough  investigation  with  refined  biochemical 
methods  will  probably  not  only  prove  the  similarity,  but  show  the  real 
reason  for  resistance  and  susceptibility. 

SUMMARY  OF  PART  II 

1.  In  making  inoculations  in  the  greenhouse  on  wheats  resistant 
to  Puccinia  graminis  tritici  it  was  found  that  only  two,  Khapli  and 
Kubanka  2094,  especially  the  former,  possessed  a  very  marked  degree 
of  real  resistance,  although  a  number  of  others  were  fairly  resistant 
in  the  field. 

2.  It  was  observed  that  the  more  resistant  a  form  proved,  the 
more  pronounced  was  the  tendency  of  the  rust  to  kill  small  areas  of 
the  leaf.     The  pustules  developed  in  these  areas  were  always  very 
small. 

3.  The  length  of  the  incubation  period  of  the  rust  is  correlated 
to  a  certain  extent  with  the  degree  of   immunity,   the  most  nearly 
immune  forms,  as  a  rule,  having  the  longest  incubation  period. 

4.  On  the  most  resistant  varieties,  such  as  Khapli,   the  spores 
are  often  small  in  size  and  sometimes  abortive. 

5.  Infection  secured  on  partially  resistant  varieties  as  a  result 
of  inoculations  with  aecidiospores   and  primary  uredospores   proved 
only  slightly  more  virulent  than  did  that  secured  by  means  of  inocula- 
tion with  long-time  uredospores. 

6.  Drought-resistant    durum    wheats    grown    in    very    dry    soil 
rusted  more  severely  than  those  grown  in  soil  with  a  higher  moisture 
content.     Minnesota  No.  163  did  not  show  much  difference,  the  plants 
in  wet  soil  being   slightly  more   severely   attacked.      The   conditions 
normal   for   the   host   plant   are   probably   also   the   conditions   under 
which  the  rust  develops  best. 

7.  It  was   found   that   in   general   the   absence   or   presence,   in 
excessive  amounts,  of  various  nutrient  substances,  such  as  nitrogen 
and  phosphorus  salts,  did  not  directly  affect  the  immunity  or  suscepti- 
bility of  wheats.     Conditions  favoring  a  normal  development  of  the 
host  were  conducive  to  vigorous  development  of  the  rust.    The  action 
of  fertilizers,  either  natural  or  artificial,  is  probably  indirect.     Tem- 
perature conditions  and  relative  humidity  of  the  atmosphere  are  prob- 
ably more  important  than  soil  conditions. 

8.  The  addition  of  copper  sulfate,  copper  carbonate,  and  iron 
sulfate  to  nutrient  media  in  which  plants  inoculated  with  rust  were 
grown  did  not  markedly  diminish  the  amount  of  rust  when  they  were 


RUST-RESISTANT  VARIETIES  OF  WHEAT  49 

used  in  such  concentration  as  to  permit  of  the  normal  development 
of  the  host  plants. 

9.  A  careful  comparison  of  the  sequence  of  infection  in  such 
a  susceptible  form  as  Minnesota  No.  163  with  that  in  such  an  immune 
form  as  Khapli  reveals  the  fact  that  the  fungus  gains  entrance  in 
the  same  manner  in  both  cases.     The  rust  mycelium  is  able  to  grow 
luxuriantly  in  Minnesota  No.  163  and  produce  spores  in  great  abun- 
dance.    In  Khapli,  however,  it  does  not  thrive.    The  reason  seems  to 
be  a  physiological  incompatibility  as  evidenced  by  the  killing  of  host 
cells  by  the  fungus  and  the  more  or  less  sudden  death  of  the  fungus 
itself.     Infection  may  occur  and  pustules  may  be  developed,  but  it  is 
evident   that   the   fungus   is   not   in   a   congenial   environment.      The 
conditions  seem  to  be  essentially  similar  when  examination  is  made  of 
a  cereal  almost  completely  immune  to  a  biologic  form,  such  as  Min- 
nesota  No.    163,   inoculated  with  Puccinia  graminis  avenae.     Here, 
however,  the  host  cells  and  rust  hyphae  are  killed  earlier  and  the  leaf 
area  involved  is  consequently  smaller.    This,  however,  requires  further 
study. 

10.  The  question  as  to  the  immediate  instruments  of  immunity 
can  probably  only  be  answered  by  means  of  biochemical  investigations. 
In  the    meantime,  morphological  and  histological    characters    being 
clearly  of  minor  importance  in  determining  immunity,  only  the  per- 
formance of  a  supposedly  resistant  variety  under  varying  conditions 
can  be  depended  on  for  a  criterion  of  its  value  in  this  respect. 

ACKNOWLEDGMENTS 

The  writer  takes  pleasure  in  making  acknowledgment  to  E.  C. 
Johnson  for  suggestions  and  material,  and  especially  to  Dr.  E.  M. 
Freeman,  under  whom  the  work  was  done,  for  many  suggestions  and 
much  criticism  during  the  progress  of  the  work. 


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EXPLANATION   OF  PLATES 

The  drawings  were  all  made  with  the  aid  of  the  camera  lucida.  The  Zeiss 
3  mm.  N.  A.  1.3  homogeneous  oil  immersion  and  compensating  ocular  No.  4 
were  used  for  all  except  Figures  8  and  10,  Plate  IX,  where  compensating  ocular 
No.  6  was  substituted. 

Plate  I  A.     Rye  after  inoculation  with  Puccinia  graminis  hordei,  showing  dead 
leaf  areas  and  a  very  few  minute  pustules. 

B.    Normal  but  rather  light  infection  on  rye  by  P.  graminis  secahs,  showing 
absence   of  dead  areas. 

Plate  II  A.    Einkorn  2433  inoculated  with  P.  graminis  originally  obtained  from 
wheat  and  confined  to  Einkorn  for  twenty-five  successive  generations. 
B.    Einkorn  2433  inoculated  with  P.  graminis  from  wheat. 

Plate  III  A.    Wheat,  Minnesota  No.  163,  inoculated  with  rust  which  had  been 
confined  to  Einkorn  for  twenty-five  generations. 

B.    Normal  infection  of  wheat  following  inoculation  with  P.  graminis  tritici 
from  wheat. 

Plate    IV  A.    Kubanka   1516  grown   in   very  wet   soil   after   inoculation   with 
P.  graminis. 
B.    Kubanka  grown  in  very  dry  soil  after  inoculation. 

Plate  V  A.    lumillo   1736  grown  in  very  wet  soil  after  inoculation   with  P. 
graminis  tritici. 

B.    The  same  variety  inoculated  under  same  condition  but  grown  in  very 
dry  soil. 

Plate  VI  A.    Khapli,  showing  characteristic  infection  following  heavy  inoculation 
with  P.  graminis. 
B.    Normal  infection  on  Minnesota  No.  163. 

Plate  VII.    Infection  of  Minnesota  No.  163  wheat  by  P.  graminis  tritici. 
1,  2  and  3 — 48  hours  after  inoculation. 
4  and  5 — 72  hours  after  inoculation. 
6  to  9 — 5  days  after  inoculation. 

1.  Surface  view  showing  appressorium  forming  over  a  stoma. 

2.  Appressorium  being  formed  directly,  without  germ  tube  development. 

3.  Germ  tube  apparently  passing  over  stoma  and  forming  a  swelling— an 

unusual  occurrence. 

4.  Part  of  a  germ  tube,  appressorium,  and  neck  connecting  the  appressorium 
with  the  substomatal  vesicle  which  has  been  cut  at  one  side. 

5.  Substomatal  vesicle,  cut  at  one  side,  beginning  to  branch. 

6.  Infection  thread  growing  from  vesicle  directly  across  substomatal  space; 

remains  of  appressorium  outside.     (Section  slightly  torn.) 

7.  Part  of  an  infection  thread  showing  haustorium  in  epidermal  cell. 
8  and  9.    Later  stages  showing  development  of  long  hyphae. 

Plate  VIII.    Infection  of  Khapli  with  P.  graminis  tritici. 
1  and  2 — 72  hours  after  inoculation. 
3  and  A — 6  days  after  inoculation. 
5,  6  and  7 — 8  days  after  inoculation. 

55 


_;  £;.  ^A  STUDY  IN  CEREAL  RUSTS 

1.  Part  of  a  germ  tube  and  an  appressorium. 

2.  Germ  tube,  appressorium,  and  substomatal  vesicle  into  which  the  nuclei 
have  passed. 

3.  Infection  thread,  with  a  few  short  branches,  killing  the  host  cell.    The 
protoplast  has  shrunk  and  the  nucleus  is  disintegrating. 

4.  Infection  thread  in  contact  with  a  cell  which  it  is  apparently  killing. 

5.  Substomatal    vesicle    and    a    number    of    somewhat     abortive     infection 
threads.    In  the  epidermal  cell  on  the  left  two  haustoria,  deeply  stained, 
and  possibly  dead.     Below,  fairly  successful  infection. 

6.  Empty  appressorium   and  vesicle.     On    the   left  infection   threads,   one 

of  which  has  sent  a  haustorium  into  an  epidermal  cell. 

7.  Infection  threads  growing  toward  leaf  tissues. 

Plate  IX.     Same  as  Plate  VIII,  except  Figs.  6  to  10,  which  are  of  Arnautka. 
1  to    5 — 10  days  after  inoculation. 
6  to  10 — 23  days  after  inoculation. 

1.  Short  hyphal  segment  containing  four  nuclei.     Haustorium  in  epidermal 
cell.     Hyphae  sent  out  from  segment  disintegrating. 

2.  Disintegrating  hypha  with  haustorium  in  host  cell. 

3.  Long  hyphae,  the  tips  of  some  becoming  much  vacuolated  and  apparently 
dying. 

4.  Typical   appearance    of    hyphae   under    epiderm,    showing   haustoria    and 
some  dying  hyphal  tips. 

5.  Hyphae  deeper  down  in  leaf  tissues  showing  tendency  to  aggregate. 

6.  Dead  host  cells  and  practically  dead  hyphae. 

7.  Hyphae  typical  of  those  in  subepidermal  wefts  showing  knoblike  branches 
which  are  often  quite  characteristic. 

8.  Small,  partly  ruptured  pustule. 

9.  Single  uredospore. 

10.     Subepidermal  weft  showing  unsuccessful  attempt   at  pustule   formation 
and  a  number  of  abortive  spores. 


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14  DAY  USE 

RETURN  TO  DESK  FROM  WHICH  BORROWED 


LD21-35m-2,'71 
(P2001slO)476 — A-32 


General  Library 

Umversity  of  California 

Berkeley 


Gaylord  Bros. 
Makers 

Syracuse,  N.  Y. 
PAT.  JAN.  21,  18 


U.C.  BERKELEY  LIBRARIES 


